Compositions, kits, and methods for identification, assessment, prevention, and therapy of endometriosis

ABSTRACT

The invention relates to newly discovered marker polypeptides associated with endometriosis. Compositions, kits, and methods for detecting, characterizing, preventing, and treating endometriosis are provided.

RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 10/887,775, filed on Jul. 9, 2004, which claimspriority from U.S. provisional patent application Ser. No. 60/575,269,filed on May 28, 2004, U.S. provisional patent application Ser. No.60/486,379, filed on Jul. 11, 2003, and from U.S. provisional patentapplication Ser. No. 60/533,430, filed on Dec. 29, 2003. The entirecontents of each of the foregoing applications are expresslyincorporated herein by reference.

BACKGROUND OF THE INVENTION

Endometriosis is a condition afflicting women of child-bearing age whichis characterized by the growth of endometrial tissue in areas outsidethe uterus. These extrauterine endometrial growths are a leading causeof pelvic pain and can also cause infertility. Endometrial growths canoccur in a variety of locations, including the lining of the pelviccavity and the outer surface of the uterus, and can also occur outsidethe abdomen, for example, in the lung.

As is the case with the uterine lining, extrauterine endometrial growthstypically respond to the varying levels of estrogen associated with themenstrual cycle. Thus, endometrial growths proceed through a cycle ofproliferation and breakdown. Unlike the uterine lining, however, thebody is unable to shed the extrauterine endometrial growths, andbreakdown of this tissue results in internal bleeding, inflammation ofthe surrounding area and formation of scar tissue. A number ofcomplications can also arise, including rupture of growths, which canspread the growths to new regions of the body, and the formation ofadhesions.

The most common symptoms of endometriosis include constant pelvic pain,infertility, low sacral backache, and heavy or irregular bleeding. Thedegree of pain does not correspond to the size or extent of endometrialgrowths, and significant pain can result even from microscopic growths.Endometrial implants can destroy ovarian and tubal tissue. Severaldisorders of menstrual cyclicity and ovulation have been suggested as abasis for the infertility caused by mild endometriosis. More subtleproblems in folliculogenesis in endometriosis patients have beenreported, including lower serum estradiol levels, smaller follicle sizeduring follicular growth, and lower oocyte fertilization rates andpregnancy rates in assisted reproduction. Problems with ovum pickup bythe fallopian tube and embryo implantation in the endometrium have alsobeen suggested.

Currently, a definitive diagnosis of endometriosis can be made only uponlaparoscopic examination of the abdomen. This is a surgical procedureperformed under local anesthesia and can indicate the extent andlocation of extrauterine endometrial growths. Laparoscopic examinationis essential because symptoms of endometriosis are similar to thesymptoms of other conditions, including ovarian cancer. Prevention ofendometriosis is not currently possible; however, treatment options areavailable based on the patient's desire for future fertility, symptoms,the stage of disease, and to some extent, age. Possible treatmentoptions include analgesic treatments, such as nonsteroidalanti-inflammatory agents and prostaglandin synthetase-inhibiting drugs,and hormonal therapy, which may be given as a means for interrupting thecycles of stimulation and bleeding of endometriotic tissue. Commonhormonal therapies include oral contraceptive pills; progestationalagents, which cause decidualization in the endometriotic tissue;danazol, a weak androgen that is the isoxazole derivative of 17α-ethinyltestosterone (ethisterone); and gonadotropin-releasing hormone (GNRH)agonists, which are analogues of the 10-amino-acid polypeptide hormoneGnRH and act via the suppression of gonadotropin secretion, resulting inelimination of ovarian steroidogenesis and suppression of endometrialimplants. Lastly, surgical treatment, including laparoscopic resection,ablation of minimal or mild endometriosis, presacral neurectomy anduterosacral ligament ablation, may be performed to excise or destroy allendometriotic tissue, remove all adhesions, and restore pelvic anatomyto the best possible condition.

Despite the treatments available for endometriosis, it would bebeneficial to provide specific non-invasive methods and reagents for thediagnosis, staging, prognosis, monitoring, and treatment ofendometriosis and endometriosis-related diseases, or to indicate a,predisposition to such for preventative measures.

SUMMARY OF THE INVENTION

The invention relates to markers (hereinafter “markers”, “markerpolypeptides” or “markers of the invention”), which are listed in Tables1-3. The invention provides nucleic acids and polypeptides that encodeor correspond to the markers (hereinafter “marker nucleic acids” and“marker polypeptides,” respectively). Tables 1-3 provide the sequenceidentifiers of the sequences of such marker peptides listed in theaccompanying Sequence Listing. The invention further providesantibodies, antibody derivatives and antibody fragments which bindspecifically with the marker polypeptides and/or fragments of the markerpolypeptides.

Table 1 lists all of the markers of the invention, whose over- orunder-abundance may be correlated with the diagnosis and prognosis ofendometriosis. In particular, Table 1 provides the name of the genecorresponding to the marker (“Gene Name”), the amino acid sequence(“Sequence”) and the sequence listing identifier of the amino acidsequence of the polypeptide marker (“SEQ ID NO (AAs)”). Table 2 listsmarkers whose over-abundance may be correlated with endometriosis ascompared to normal samples from control subjects that do not haveendometriosis. Table 3 lists markers whose under-abundance may becorrelated with endometriosis as compared to normal samples from controlsubjects that do not have endometriosis.

The invention also relates to various methods, reagents and kits fordiagnosing, staging, prognosticating, monitoring and treatingendometriosis. “Endometriosis” as used herein includes a disorder inwhich abnormal growth of tissue, histologically resembling theendometrium, is present in locations other than the uterine lining (seeAinbinder et al., Current Obstetric & Gynecologic Diagnosis & Treatment,9^(th) ed., Lange Medical Books/McGraw-Hill, 2003). As used herein, an“endometriosis-related disease” (also referred to herein as a “disorder”or “condition”) may include a disease, disorder, or condition, whoseonset was related to endometriosis. Such diseases, disorders andconditions include infertility, and abdominal and/or pelvic pain.

In one embodiment, the invention provides a diagnostic method ofassessing whether a patient has endometriosis or has higher than normalrisk for developing endometriosis, comprising the steps of comparing theabundance of a marker of the invention in a patient sample and thenormal abundance of the marker in a control, e.g., a sample from asubject that does not have endometriosis. A difference in the abundanceof the marker in the patient sample, as compared to the normalabundance, is an indication that the patient is afflicted withendometriosis or has higher than normal risk for developingendometriosis.

The methods of the present invention can be of use in identifyingpatients having an enhanced risk of developing endometriosis (e.g.,patients having a familial history of endometriosis and patientsidentified as having altered abundance of a marker of the invention).The methods of the present invention may further be of particular use inevaluating the specific stage of endometriosis, as well as in assessingthe progression of the disease. The methods of the present invention arealso useful in predicting the clinical outcome for a patient withendometriosis, or for a patient who has undergone therapy to eradicateendometriosis. The methods of the present invention are also useful inassessing the efficacy of treatment of a patient diagnosed withendometriosis (e.g., the efficacy of hormonal suppression or surgicalablation of endometrial implants).

The markers of the invention set forth in Table 1 may be used in themethods of the invention. It will be appreciated that in the methods ofthe invention, over-abundance of the markers set forth in Table 2 in thepatient sample may be correlated with endometriosis as compared tonormal samples from control subjects. Likewise, in the methods of theinvention, under-abundance of the markers set forth in Table 3 in thepatient sample may be correlated with endometriosis as compared tonormal samples from control subjects.

In a preferred diagnostic method of assessing whether a patient isafflicted with endometriosis (e.g., new detection (“screening”),detection of recurrence), the method comprises comparing:

-   -   a) the abundance of a marker listed in Table 1 in a sample from        the patient, and    -   b) the normal abundance of the marker.        A different abundance of the marker in the patient sample, as        compared to the level in the control subject, i.e., increased or        decreased as specified in Tables 2-3, is an indication that the        patient is afflicted with endometriosis.

The invention additionally provides a diagnostic method for assessingthe aggressiveness of endometriosis, the method comprising comparing:

-   -   a) the abundance of a marker listed in Table 1 in a sample from        the patient, and    -   b) the normal abundance of the marker.        A different abundance in the patient sample, as compared to the        normal level, i.e., increased or decreased as specified in        Tables 2-3, is an indication that the patient has an aggressive        form of endometriosis or is likely to develop endometriosis.

The invention also provides methods for assessing the efficacy of atherapy for inhibiting endometriosis in a patient. Such methods comprisecomparing:

-   -   a) the abundance of a marker of the invention in a first sample        obtained from the patient prior to providing at least a portion        of the therapy to the patient, and    -   b) the abundance of the marker in a second sample obtained from        the patient following provision of the portion of the therapy.        An altered abundance of the marker in the second sample relative        to that in the first sample, i.e., increased or decreased as        specified in Tables 2-3, is an indication that the therapy is        efficacious for inhibiting endometriosis in the patient.

It will be appreciated that in the methods of the present invention, the“therapy” may be any therapy for treating endometriosis including, butnot limited to, analgesic treatments, hormone therapy, surgical removalof endometrial implants, gene therapy and biologic therapy such as theadministering of antibodies. Thus, the methods of the invention may beused to evaluate a patient before, during and after therapy, forexample, to evaluate the reduction in endometriosis due to the therapy.

In a preferred embodiment, the methods are directed to therapy using achemical or biologic agent. These methods comprise comparing:

-   -   a) the abundance of a marker of the invention in a first sample        obtained from the patient and maintained in the presence of the        chemical or biologic agent, and    -   b) the abundance of the marker in a second sample obtained from        the patient and maintained in the absence of the agent.        An altered abundance of the marker in the second sample relative        to that in the first sample, i.e., increased or decreased as        specified in the above-described methods and in Tables 2-3, is        an indication that the agent is efficacious for inhibiting        endometriosis, in the patient. In one embodiment, the first and        second samples can be portions of a single sample obtained from        the patient or portions of pooled samples obtained from the        patient.

The invention additionally provides a monitoring method for assessingthe progression of endometriosis in a patient, the method comprising:

-   -   a) detecting in a sample from the patient at a first time point,        the abundance of a marker of the invention;    -   b) repeating step a) at a subsequent time point in time; and    -   c) comparing the abundance detected in steps a) and b), and        therefrom monitoring the progression of endometriosis in the        patient.        A different abundance of the marker in the sample at the        subsequent time point from that of the sample at the first time        point, i.e., increased or decreased as specified in the        above-described methods and in Tables 2 and 3, is an indication        that the endometriosis has progressed or regressed in the        patient.

The invention moreover provides a test method for selecting acomposition for inhibiting endometriosis in a patient. This methodcomprises the steps of:

-   -   a) obtaining a sample from the patient;    -   b) separately maintaining aliquots of the sample in the presence        of a plurality of test compositions;    -   c) comparing the abundance of a marker of the invention in each        of the aliquots; and    -   d) selecting one of the test compositions which significantly        alters the abundance of the marker in the aliquot containing        that test composition, relative to the abundance of the marker        in the presence of the other test compositions.

In addition, the invention further provides a method of inhibitingendometriosis in a patient. This method comprises the steps of:

-   -   a) obtaining a sample from the patient;    -   b) separately maintaining aliquots of the sample in the presence        of a plurality of compositions;    -   c) comparing the abundance of a marker of the invention in each        of the aliquots; and    -   d) administering to the patient at least one of the compositions        which significantly alters the abundance of the marker in the        aliquot containing that composition, relative to the abundance        of the marker in the presence of the other compositions.

In the aforementioned methods, the samples or patient samples comprisecells, tissues and/or fluids obtained from the patient. The cells may befound in a cervical smear collected, for example, by a cervical brush.In another embodiment, the sample is a body fluid. Such fluids include,for example, blood fluids, serum, plasma, a blood fraction, lymph,ascitic fluids, gynecological fluids, urine, peritoneal fluid,cerebrospinal fluid, and fluids collected by vaginal rinsing. In afurther embodiment, the patient sample is in vivo.

According to the invention, the abundance of a marker of the inventionin a sample can be assessed, for example, by detecting the presence inthe sample of:

-   -   the marker polypeptide (e.g., a polypeptide having one of the        sequences of SEQ ID NO (AAs)) or a fragment of the polypeptide        (e.g. by using a reagent, such as an antibody, an antibody        derivative, an antibody fragment or single-chain antibody, which        binds specifically with the protein or protein fragment, or via        chromatography with a suitable detector)    -   a metabolite which is produced directly (i.e., catalyzed) or        indirectly by the corresponding marker polypeptide; or    -   a metabolite of the marker polypeptide.

According to the invention, any of the aforementioned methods may beperformed using a plurality (e.g. 2, 3, 5, or 10 or more) of markers ofthe invention, optionally in combination with endometriosis markersknown in the art. In such methods, the abundance within the sample ofeach of a plurality of markers, at least one of which is a marker of theinvention, is compared with the normal abundance of each of theplurality of markers. A significantly altered (i.e., increased ordecreased as specified in the above-described methods using a singlemarker) abundance in the sample of one or more markers of the invention,or some combination thereof, relative to that marker's correspondingnormal or control level, is an indication that the patient is afflictedwith endometriosis.

In a further aspect, the invention provides an antibody, an antibodyderivative, or an antibody fragment, which binds specifically with amarker polypeptide (e.g., a polypeptide having the sequence of any ofthe SEQ ID NO (AAs)) or a fragment of the polypeptide. The inventionalso provides methods for making such an antibody, antibody derivative,and antibody fragment. Such methods may comprise immunizing a mammalwith a polypeptide comprising the entirety, or a fragment, preferably asegment of 7 or more, more preferably 10 or more, amino acids, of amarker polypeptide (e.g., a polypeptide having the sequence of any ofthe SEQ ID NO (AAs)), wherein the polypeptide may be obtained from acell or by chemical synthesis. The methods of the invention alsoencompass producing monoclonal and single-chain antibodies, which wouldfurther comprise isolating splenocytes from the immunized mammal, fusingthe isolated splenocytes with an immortalized cell line to formhybridomas, and screening individual hybridomas for those that producean antibody that binds specifically with a marker polypeptide or afragment of the polypeptide.

In another aspect, the invention relates to various diagnostic and testkits. In one embodiment, the invention provides a kit for assessingwhether a patient is afflicted with endometriosis. The kit comprises areagent for assessing the abundance of a marker or a plurality ofmarkers of the invention. In another embodiment, the invention providesa kit for assessing the suitability of a chemical or biologic agent forinhibiting endometriosis in a patient. Such a kit comprises a reagentfor assessing the abundance of a marker of the invention, and may alsocomprise one or more additional such agents. Such kits comprise anantibody, an antibody derivative, or an antibody fragment, which bindsspecifically with a marker polypeptide, or a fragment of thepolypeptide. Such kits may also comprise a plurality of antibodies,antibody derivatives, or antibody fragments wherein the plurality ofsuch antibody agents binds specifically with a marker polypeptide, or afragment of the marker polypeptide. In another embodiment, the kitincludes one or more synthetic standards and/or internal standards.

It will be appreciated that the methods and kits of the presentinvention may also include known endometriosis markers. It will furtherbe appreciated that the methods and kits may be used to identifyconditions related to endometriosis, such as infertility or abdominaland/or pelvic pain of unknown ideology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-6 depict scatter plots showing the concentrations (given ascounts) of a given marker in the serum of women diagnosed withendometriosis, the serum of healthy women, and, in certain cases, theserum of women diagnosed with PCOS or PID.

FIGS. 1 and 2 demonstrate that certain markers of the invention,including fibrinopeptide B derivative (SEQ ID NO:28), full lengthfibrinopeptide A (SEQ ID NO:3) and fibrinopeptide A fragment (SEQ IDNO:7), are present in higher concentrations in the serum of patientsdiagnosed with endometriosis as compared to healthy patients that do nothave endometriosis.

FIG. 3 demonstrates that a marker of the invention, the fibrinopeptide Aderivative (SEQ ID NO:4), is present at higher concentrations in theserum of patients diagnosed with endometriosis as compared to serum ofhealthy patients that do not have endometriosis or patients diagnosedwith polycystic ovarian syndrome (PCOS) and pelvic inflammatory disease(PID).

FIG. 4 demonstrates that certain markers of the invention, thymosinfragments (SEQ ID NOS:42 and 43), are present at lower concentrations inthe serum of patients diagnosed with endometriosis, PCOS and PID ascompared to serum of healthy patients that do not have endometriosis.

FIG. 5 demonstrates that a marker of the invention, phosphoserinefibrinopeptide A (SEQ ID NO:22), is present at lower concentrations inthe serum of healthy patients as compared to serum of patients diagnosedwith endometriosis, PCOS or PID.

FIG. 6 demonstrates that a marker of the invention, the internalfibrinogen alpha fragment (SEQ ID NO:29), is present at lowerconcentrations in the serum of patients diagnosed with endometriosis ascompared to healthy patients that do not have endometriosis, PCOS orPID.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to newly discovered markers, whose expression iscorrelated with the disease state of endometriosis. In particular, thepresent invention relates to the discovery that patient samples, e.g.,serum samples, from women who have endometriosis have higher levels ofpolypeptides derived from fibrinogen proteolysis than women who do nothave endometriosis, as shown either through an absence of signs andsymptoms of endometriosis or by another diagnostic method, such aslaparoscopy. It has now been found that, compared to women who do nothave endometriosis, patient samples, e.g., serum samples, from women whohave endometriosis have higher levels of certain markers of theinvention and lower levels of certain other markers of the invention.Thus, it has been discovered that for certain markers of the inventionan altered, e.g., significantly higher than normal or significantlylower than normal abundance of these markers, or combination of thesemarkers, correlates with the presence of endometriosis in a patient.Methods are provided for detecting the presence or absence ofendometriosis in a sample, the stage and progression of endometriosis,predicting the likely clinical outcome of a patient diagnosed withendometriosis, and other characteristics of endometriosis that arerelevant to prevention, diagnosis, characterization, and therapy ofendometriosis in a patient. Methods of treating endometriosis are alsoprovided.

Table 1 lists all of the markers of the invention, whose over- orunder-abundance correlates with endometriosis as compared to normalsamples from patients that do not have endometriosis. Table 2 listsmarkers whose over-expression correlates with endometriosis as comparedto normal samples from control subjects. Table 3 lists markers whoseunder-expression correlates with endometriosis as compared to normalsamples from control subjects.

In one embodiment, a marker of the invention is fibrinogen, such as thefibrinogen a (SEQ ID NO:1) or β-chain (SEQ ID NO:2) or a fragmentthereof, such as fibrinopeptide A (SEQ ID NO:3), fibrinopeptide B (SEQID NO:27) or a fragment thereof (SEQ ID NO:28). The fragment offibrinopeptide A can be, for example, a fragment derived from N-terminaltruncation of fibrinopeptide A (SEQ ID NOS:5-21). Furthermore, themarkers of the invention include fibrinopeptide A and fragments thereof,wherein the serine residue has been converted into dehydroalanine (SEQID NO:4) or in which the serine residue is phosphorylated (SEQ ID NOS:22to 26). In another embodiment, the markers of the invention includefibrinopeptide B (SEQ ID NO:27) and derivatives and fragments thereof,including des-arginine-L-pyroglutamic acid fibrinopeptide B (SEQ IDNO:28). In a further aspect, the markers of the invention include aninternal fragment of the fibrinogen a chain, such as a polypeptidehaving the sequence set forth in (SEQ ID NO:29).

The markers of the invention also include polypeptides derived fromComplement component 3 (Complement C3) (SEQ ID NO:30). Complement C3 isconverted by the enzyme C3 convertase to two protein products, C3a (SEQID NO:31) and C3b (SEQ ID NO:32). C3b is in turn converted by thrombinto the product polypeptides iC3b (SEQ ID NO:33) and C3f (SEQ ID NO:36).In one embodiment, the markers of the invention include polypeptideswhich are N-terminal fragments of iC3b, such as polypeptides having thesequence set forth in SEQ ID NOS:34 and 35. The markers of the inventionmay also include full length C3f (SEQ ID NO:36) and fragments thereof,including polypeptides having the sequence set forth as (SEQ ID NO:37).In yet another embodiment, the markers of the invention includepolypeptides derived from thymosin beta 1 (SEQ ID NO:38), thymosin beta3 (SEQ ID NO:39), thymosin beta 4 (SEQ ID NO:40), or thymosin beta 6(SEQ ID NO:41), and fragments thereof, including polypeptides having thesequence set forth as SEQ ID NOS:42 and 43. The invention also providesisolated nucleic acid molecules which encode the amino acid sequencesset forth herein.

TABLE 1 SEQ ID NO Gene Name Exemplary Sequence (AAs) Fibrinogen (alphachain) GenBank Accession No. P02671  1 Fibrinogen (beta chain) GenBankAccession No. P02675  2 Fibrinopeptide A H-ADSGEGDFLAEGGGVR-OH  3Fibrinopeptide A H-AD(dehydroA)GEGDFLAEGGGVR-OH  4 (wherein serineresidue has been converted into dehydroalanine) a fragment derived fromH-DSGEGDFLAEGGGVR-QH;  5 an N-terminal H-DSGEGDFLAEGGGV-OH;  6truncation of H-SGEGDFLAEGGGVR-OH;  7 fibrinopeptide AH-SGEGDFLAEGGGV-OH;  8 H-GEGDFLAEGGGVR-OH;  9 H-GEGDFLAEGGGV-OH; 10H-EGDFLAEGGGVR-OH; 11 H-EGDFLAEGGGV-OH; 12 H-GDFLAEGGGVR-OH; 13H-GDFLAEGGGV-OH; 14 H-DFLAEGGGVR-OH; 15 H-DFLAEGGGV-OH; 16H-FLAEGGGVR-OH; 17 H-FLAEGGGV-OH; 18 H-LAEGGGV-OH; 19 H-AEGGGV-OH; 20H-EGGGV-OH. 21 Phosphoserine- H-ADS*GEGDFLAEGGGVR-OH 22 containing (S*= phosphoserine) fibrinopeptide A Phosphoserine- H-DS*GEGDFLAEGGGVR-OH;23 containing H-DS*GEGDFLAEGGGV-OH; 24 fibrinopeptide AH-S*GEGDFLAEGGGVR˜OH; 25 fragments derived H-S*GEGDFLAEGGGV-OH 26 froman N-terminal (S* = phosphoserine) truncation thereof fibrinopeptide BQGVNDNEEGFFSAR 27 des-arginine-L- pyr-EGVNDNEEGFFSA-OH 28 pyroglutamicacid fibrinopeptide B internal fragment H-DEAGSEADHEGTHST-OH 29 of thefibrinogen alpha chain Complement C3 GenBank Accession No. P01024 30 C3aSVQLTEKRM DKVGKYPKEL RKCCEDGMRE 31 NPMRFSCQRR TRFISLGEAC KKVFLDCCNYITELRRQHAR ASHLGLAR C3b See Sequence Listing 32 iC3b See SequenceListing 33 N-terminal fragments H-SEETKENEGFTVTAEG-OH; 34 of iC3bH-EETKENEGFTVTAEG-OH 35 C3f H-SSKITHRIHWESASLLR-OH 36 C3f fragmentH-HWESASLL-OH 37 Thymosin beta 1 SDKPDMAEME KFDKSKLKKT ETQEKNPLPS 38KETIEQEKQA GES Thymosin beta 3 SDKPDMAEIE KFDKPKLKKT ETQEKNPLPS 39KETIEQEKQA GES Thymosin beta 4 GenBank Accession No. P01253 40 Thymosinbeta 6 SDKSDMAEIE KFDKSKLKKT ETQEKNPLPS 41 KETIEQEKQA GES Fragments ofthymosin H-TQEKNPLPSKETIEQEKQAGES-OH; 42 Pyr-EKNPLPSKETIEQEKQAGES-OH 43

TABLE 2 SEQ ID NO Gene Name Exemplary Sequence (AAs) Fibrinogen (alphachain) GenBank Accession No. P02671  1 Fibrinogen (beta chain) GenBankAccession No. P02675  2 Fibrinopeptide A H-ADSGEGDFLAEGGGVR-OH  3Fibrinopeptide A (wherein H-AD(dehydroA)GEGDFLAEGGGVR-OH  4 serineresidue has been converted into dehydroalanine) a fragment derived fromH-DSGEGDFLAEGGGVR-OH;  5 an N-terminal truncation H-DSGEGDFLAEGGGV-OH; 6 of fibrinopeptide A H-SGEGDFLAEGGGVR-OH;  7 H-SGEGDFLAEGGGV-OH;  8H-GEGDFLAEGGGVR-OH;  9 H-GEGDFLAEGGGV-OH; 10 H-EGDFLAEGGGVR-OH; 11H-EGDFLAEGGGV-OH; 12 H-GDFLAEGGGVR-OH; 13 H-GDFLAEGGGV-OH; 14H-DFLAEGGGVR-OH; 15 H-DFLAEGGGV-OH; 16 H-FLAEGGGVR-OH; 17 H-FLAEGGGV-OH;18 H-LAEGGGV-OH; 19 H-AEGGGV-OH; 20 H-EGGGV-OH 21Phosphoserine-containing H-ADS*GEGDFLAEGGGVR-OH 22 fibrinopeptide A (S*= phosphoserine) Phosphoserine-containing H-DS*GEGDFLAEGGGVR-OH; 23fibrinopeptide A fragments H-DS*GEGDFLAEGGGV-OH; 24 derived from anN-terminal H-S*GEGDFLAEGGGVR-OH; 25 truncation thereofH-S*GEGDFLAEGGGV-OH 26 (S* = phosphoserine) fibrinopeptide BQGVNDNEEGFFSAR 27 des-arginine-L- Pyr-EGVNDNEEGFFSA-OH 28 pyroglutamicacid fibrinopeptide B internal fragment of the H-DEAGSEADHEGTHST-OH 29fibrinogen alpha chain Complement C3 GenBank Accession No. P01024 30 C3aSVQLTEKRM DKVGKYPKEL RKCCEDGMRE 31 NPMRFSCQRR TRFISLGEAC KKVFLDCCNYITELRRQHAR ASHLGLAR C3b See Sequence Listing 32 C3fH-SSKITHRIHWESASLLR-OH 36 C3f fragment H-HWESASLL-OH 37

TABLE 3 SEQ ID NO Gene Name Exemplary Sequence (AAs) iC3b See SequenceListing 33 N-terminal fragments of iC3b H-SEETKENEGFTVTAEG-OH; 34H-EETKENEGFTVTAEG-OH 35 Thymosin beta 1 SDKPDMAEME KFDKSKLKKT ETQEKNPLPS38 KETIEQEKQA GES Thymosin beta 3 SDKPDMAEIE KFDKPKLKKT ETQEKNPLPS 39KETIEQEKQA GES Thymosin beta 4 GenBank Accession No. P01253 40 Thymosinbeta 6 SDKSDMAEIE KFDKSKLKKT ETQEKNPLPS 41 KETIEQEKQA GES Fragments ofH-TQEKNPLPSKETIEQEKQAGES-OH; 42 thymosin Pyr-EKNPLPSKETIEQEKQAGES-OH 43

Thus, in one embodiment, a marker polypeptide of the invention isfibrinogen or a fragment thereof, such as fibrinopeptide A or a fragmentthereof. The fragment of fibrinopeptide A can be, for example, afragment derived from an N-terminal truncation of fibrinopeptide A. Forexample, the marker polypeptides of the invention can include one ormore polypeptides having the sequences ADSGEGDFLAEGGGVR (fibrinopeptideA) (SEQ ID NO:3); DSGEGDFLAEGGGVR (SEQ ID NO:5); DSGEGDFLAEGGGV (SEQ IDNO:6); SGEGDFLAEGGGVR (SEQ ID NO:7); SGEGDFLAEGGGV (SEQ ID NO:8);GEGDFLAEGGGVR (SEQ ID NO:9); GEGDFLAEGGGV (SEQ ID NO:10); EGDFLAEGGGVR(SEQ ID NO:11); EGDFLAEGGGV (SEQ ID NO:12); GDFLAEGGGVR (SEQ ID NO:13);GDFLAEGGGV (SEQ ID NO:14); DFLAEGGGVR (SEQ ID NO:15); DFLAEGGGV (SEQ IDNO:16); FLAEGGGVR (SEQ ID NO:17); FLAEGGGV (SEQ ID NO:18); LAEGGGV (SEQID NO:19); AEGGGV (SEQ ID NO:20); and EGGGV (SEQ ID NO:21).Fibrinopeptide A and fragments thereof wherein the serine residue hasbeen phosphorylated or converted into dehydroalanine are also included.Such peptides include AD(dehydroA)GEGDFLAEGGGVR (SEQ ID NO:4);ADS*GEGDFLAEGGGVR (phosphorylated fibrinopeptide A) (SEQ ID NO:22);DS*GEGDFLAEGGGVR (SEQ ID NO:23); DS*GEGDFLAEGGGV (SEQ ID NO:24);S*GEGDFLAEGGGVR (SEQ ID NO:25); and S*GEGDFLAEGGGV (SEQ ID NO:26), where“dehydroA” represents dehydroalanine and “S*” represents phosphoserine.

The marker polypeptides of the invention also include the fibrinogenβ-chain or a fragment thereof, such as fibrinopeptide B or a fragmentthereof. In another embodiment, the marker polypeptides of the inventioninclude fibrinopeptide B and derivatives and fragments thereof,including des-arginine-L-pyroglutamic acid fibrinopeptide B,pyr-EGVNDNEEGFFSA (SEQ ID NO:28). The N-terminal Gln has been cyclizedto form a pyroglutamate. It will be appreciated that pyr-E in thesequences of the invention represents pyroglutamate.

In a further aspect, the marker polypeptides of the invention include aninternal fragment of the fibrinogen α-chain, such as a peptide havingthe sequence DEAGSEADHEGTHST (SEQ ID NO:29).

The marker polypeptides of the invention also include peptides derivedfrom Complement component 3 (Complement C3). Complement C3 is convertedby the enzyme C3 convertase to two protein products, C3a and C3b. C3b isin turn converted by thrombin to the product peptides iC3b and C3f. Inone embodiment, the biomarkers of the invention include peptides whichare N-terminal fragments of iC3b, such as peptides having the sequenceSEETKENEGFTVTAEG (SEQ ID NO:34) or EETKENEGFTVTAEG (SEQ ID NO:35). Thebiomarkers of the invention also include full length C3f, having theamino acid sequence SSKITHRIHWESASLLR (SEQ ID NO:36), and fragmentsthereof, including peptides having the sequence HWESASLL (SEQ ID NO:37).

In yet another embodiment, the marker polypeptides of the inventioninclude peptides derived from thymosin beta 1, thymosin beta 3, thymosinbeta 4, or thymosin beta 6, and fragments thereof, including peptideshaving the sequence TQEKNPLPSKETIEQEKQAGES (SEQ ID NO:42) or thesequence pyr-EKNPLPSKETIEQEKQAGES (SEQ ID NO:43). It will be appreciatedthat pyr-E in the sequences of the invention represents pyroglutamate.

Unless otherwise indicated, the marker polypeptides of the inventionhave free N- and C-termini. This is shown in Tables 1 to 3 by theN-terminal “H-”, which indicates a free amino group at the N-terminus,and the C-terminal “—OH”, which indicates a free carboxyl group at theC-terminus.

In another embodiment, the present invention provides isolatedpolypeptides which are fragments of fibrinogen, fibrinogen α-chain,fibrinopeptide A, fibrinogen β-chain, fibrinopeptide B, C3a, C3b, iC3b,C3f, thymosin beta 1, thymosin beta 3, thymosin beta 4, thymosin beta 6or analogues thereof. The isolated polypeptides of the invention includepolypeptides having an amino acid sequence selected from the groupconsisting of ADSGEGDFLAEGGGVR (fibrinopeptide A) (SEQ ID NO:3);DSGEGDFLAEGGGVR (SEQ ID NO:5); DSGEGDFLAEGGGV (SEQ ID NO:6);SGEGDFLAEGGGVR (SEQ ID NO:7); SGEGDFLAEGGGV (SEQ ID NO:8); GEGDFLAEGGGVR(SEQ ID NO:9); GEGDFLAEGGGV (SEQ ID NO: 10); EGDFLAEGGGVR (SEQ IDNO:11); EGDFLAEGGGV (SEQ ID NO:12); GDFLAEGGGVR (SEQ ID NO:13);GDFLAEGGGV (SEQ ID NO:14); DFLAEGGGVR (SEQ ID NO:15); DFLAEGGGV (SEQ IDNO:16); FLAEGGGVR (SEQ ID NO:17); FLAEGGGV (SEQ ID NO:18); LAEGGGV (SEQID NO:19); AEGGGV (SEQ ID NO:20); EGGGV (SEQ ID NO:21);D(dehydroA)GEGDFLAEGGGVR (SEQ ID NO:4); ADS*GEGDFLAEGGGVR(phosphorylated fibrinopeptide A) (SEQ ID NO:22); DS*GEGDFLAEGGGVR (SEQID NO:23); DS*GEGDFLAEGGGV (SEQ ID NO:24); S*GEGDFLAEGGGVR (SEQ IDNO:25); S*GEGDFLAEGGGV (SEQ ID NO:26); pyr-EGVNDNEEGFFSA (SEQ ID NO:28);DEAGSEADHEGTHST (SEQ ID NO:29); SEETKENEGFTVTAEG (SEQ ID NO:34);EETKENEGFTVTAEG (SEQ ID NO:35); SSKITHRIHWESASLLR (SEQ ID NO:36);HWESASLL (SEQ ID NO:37); TQEKNPLPSKETIEQEKQAGES (SEQ ID NO:42) andpyr-EKNPLPSKETIEQEKQAGES (SEQ ID NO:43).

The invention also provides isolated nucleic acid molecules which encodean amino acid sequence set forth herein.

In another embodiment, the invention relates to antibodies that bind toand/or can detect a polypeptide of the invention. The antibodies can bepolyclonal antibodies or monoclonal antibodies, humanized or chimericantibodies or fragments thereof.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The “abundance” of a polypeptide marker in a sample is the amount of themarker in the sample, as determined by quantitative analysis. Theabundance can be expressed as the absolute amount of the markerpolypeptide within the sample, or as a relative amount, such as amountof the polypeptide per unit mass of the sample or the concentration ofthe polypeptide within the sample. The abundance of the polypeptide inthe sample can depend upon a number of factors, such as the level ofexpression of the polypeptide within the tissue sampled, the extent oftransport of the polypeptide from its source to the tissue sampled andthe extent of metabolism or processing of a parent protein to producethe polypeptide.

An “analogue” of a reference polypeptide is a polypeptide havingsubstantial identity to the reference polypeptide. For example, ananalogue can exhibit 70%, 75%, 80%, 85%, 90% or 95% identity to thereference polypeptide. The analogue can also be a truncated polypeptideresulting from C- and/or N-terminal truncation of the referencepolypeptide. An analogue can also have one or more amino acidsubstitutions compared to the reference polypeptide, such as thesubstitution of one or more residues with another naturally occurringL-configuration residue, or a non-natural amino acid residue, such as aD-configuration residue or a D- or L-configuration residue bearing aside chain which is different from the side chains of the twentynaturally-occurring L-amino acids. In one embodiment, the analogueresults from conservative substitution of one or more residues in thereference polypeptide. The term “conservative substitution” is known inthe art and relates to the substitution of an amino acid residue with aresidue bearing a side chain of similar properties. In a preferredembodiment, an analogue of a reference polypeptide is immunologicallycross-reactive with the reference polypeptide, that is, antibodiesraised to the analogue are able to bind to and/or detect the referencepolypeptide.

A “marker”, as this term is used herein, is a polypeptide whose alteredabundance in a tissue, cell or bodily fluid compared to its abundance innormal or healthy tissue, cell or bodily fluid is associated with adisease state, such as endometriosis. A “marker nucleic acid” is anucleic acid (e.g., mRNA, cDNA) which encodes a marker of the invention.Such marker nucleic acids include DNA (e.g., cDNA) encoding the entireor a partial sequence of any of the SEQ ID NO (AA) or the complement ofsuch a sequence. The marker nucleic acids also include RNA correspondingto the entire or a partial sequence of any marker nucleic acid sequenceor the complement of such a sequence, wherein all thymidine residues arereplaced with uridine residues.

The term “probe” refers to any molecule which is capable of selectivelybinding to a specifically intended target molecule, for example, apolynucleotide, a polynucleotide transcript or a polypeptide. Probes canbe either synthesized by one skilled in the art, or derived fromappropriate biological preparations. For purposes of detection of thetarget molecule, probes may be specifically designed to be labeled, asdescribed herein. Examples of molecules that can be utilized as probesinclude, but are not limited to, RNA, DNA, proteins, antibodies, andorganic molecules.

As used herein, a “patient sample” or “bodily fluid” can be any bodilyfluid in which differences in the amount of at least one marker of theinvention is indicative of the presence of endometriosis. Suitablebodily fluids include blood, a blood fraction, urine, saliva, tears, andcerebrospinal fluid. In a preferred embodiment, the bodily fluid isblood or a fraction thereof, such as serum or plasma. More preferably,the bodily fluid is serum. An “endometrial-associated fluid” is a fluidwhich, when in the body of a patient, contacts or passes through theendometrium or into which cells, nucleic acids or proteins are shed fromendometrial cells. Exemplary endometrial-associated body fluids includeperitoneal fluid, which is a fluid obtained from the peritoneal cavity,the resulting fluids obtained from a PAP smear procedure, gynecologicalfluids, and fluids collected by vaginal rinsing.

The term “normal abundance” of a marker is the abundance of the markerin a particular sample, such as blood serum, which is indicative of theabsence of endometriosis. For example, the normal abundance of a markeris the abundance found in a sample from a control subject not afflictedwith endometriosis. Preferably, the normal abundance is an average valueor a range of values found by analysis of samples derived from aplurality of control subjects not afflicted with endometriosis.

The terms “significantly greater abundance” and “overabundance” of amarker refers to an amount of the marker in a test sample that isgreater than the normal abundance of the marker by an amount equal to atleast the standard error of the assay employed to determine abundance,and is preferably at least twice, the normal abundance of the marker.

The terms “significantly lower abundance” and “under-abundance” of amarker refer to an amount of the marker in a test sample that is that isless than the normal abundance of the marker by an amount equal to atleast the standard error of the assay employed to determine abundance,and is preferably 50% or less of the normal abundance of the marker.

The term “altered abundance” refers to a significantly greater abundanceor a significantly lower abundance when compared to the normalabundance.

When comparing the abundance of a marker in a subject to the normalabundance, the comparison is made using like samples. For example, theabundance of a given marker in a serum sample derived from a subject iscompared to the normal abundance of the marker in serum. Thus, a markerwhich is over-abundant or under-abundant in the serum of a subject hasan altered abundance relative to the normal abundance in serum.

As used herein, a “patient,” “subject” or “female subject” can be anyfemale mammal, and is preferably a human female. More preferably, thesubject is a human female of child-bearing age, i.e., a post-pubescenthuman female who has not yet entered menopause. The woman may beselected by a physician for evaluation by the method of the invention onthe basis of the presence of one or more symptoms of endometriosis, suchas abdominal or pelvic pain, irregular menstruation, or infertility.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue-specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cell under mostor all physiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

A chemical entity, such as a protein, polypeptide or antibody, is“isolated” if a composition comprising the entity is substantially freeof other macromolecules, such as other proteins. A chemical entity is“purified” in a composition in which the entity is present insubstantially greater relative concentration than it exists in itsnatural state, for example in a body fluid of a subject. Preferably thechemical entity comprises at least 80%, more preferably at least 90%,and even more preferably at least 95% by weight of the macromolecularspecies present in the composition. Most preferably, the chemical entityis purified to homogeneity, i.e., other macromolecular species are notsignificantly detectable using standard techniques, such aspolyacrylamide gel electrophoresis and high performance liquidchromatography.

A “transcribed polynucleotide” or “nucleotide transcript” is apolynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA orcDNA) which is complementary to or homologous with all or a portion of amature mRNA made by transcription of a marker of the invention andnormal post-transcriptional processing (e.g. splicing), if any, of theRNA transcript, and reverse transcription of the RNA transcript.

“Homologous” as used herein, refers to amino acid sequence similaritybetween regions of two different polypeptide sequences. When an aminoacid residue position in both regions is occupied by the same amino acidresidue, then the regions are homologous at that position. A firstregion is homologous to a second region if at least one amino acidresidue position of each region is occupied by the same residue.Homology between two regions is expressed in terms of the proportion ofamino acid residue positions of the two regions that are occupied by thesame amino acid residue. Preferably, the first region comprises a firstportion and the second region comprises a second portion, whereby, atleast about 50%, and preferably at least about 75%, at least about 90%,or at least about 95% of the amino acid residue positions of each of theportions are occupied by the same amino acid residue.

A molecule is “fixed” or “affixed” to a substrate if it is covalently ornon-covalently associated with the substrate such the substrate can berinsed with a fluid (e.g. standard saline citrate, pH 7.4) without asubstantial fraction of the molecule dissociating from the substrate.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs in anorganism found in nature.

The term “synthetic standard” refers to a synthetic or recombinantpolypeptide which is structurally equivalent to one of the markerpolypeptides of the invention. The term “internal standard” refers to acompound which is chemically and structurally similar to a markerpolypeptide, but which differs from the marker polypeptide in mass.

Endometriosis is “inhibited” if at least one symptom of the disease isalleviated, terminated, slowed, or prevented. As used herein,endometriosis is also “inhibited” if recurrence or progression of thedisease is reduced, slowed, delayed, or prevented.

A kit is any manufacture (e.g. a package or container) comprising atleast one reagent, e.g. a probe, an antibody, a synthetic standard or aninternal standard, for specifically determining the abundance of amarker of the invention. The kit may be promoted, distributed, or soldas a unit for performing the methods of the present invention.

“Polypeptides of the invention” encompass marker polypeptides, such asthe polypeptides set forth in Table 1 and their fragments; variantmarker polypeptides, such as polypeptides which are homologous to thepolypeptides set forth in Table 1, for example, polypeptides which canbe used as internal standards, and their fragments; polypeptides andpolypeptides comprising an at least 7, 10 or 15 amino acid segment of amarker or variant marker polypeptide; and fusion proteins comprising amarker or variant marker polypeptide, or an at least 7, 10 or 15 aminoacid segment of a marker or variant marker polypeptide.

Unless otherwise specified herein, the terms “antibody” and “antibodies”broadly encompass naturally-occurring forms of antibodies (e.g., IgG,IgA, IgM, IgE) and recombinant antibodies such as single-chainantibodies, chimeric and humanized antibodies and multi-specificantibodies, as well as fragments and derivatives of all of theforegoing, which fragments and derivatives have at least an antigenicbinding site. Antibody derivatives may comprise a protein or chemicalmoiety conjugated to an antibody.

DESCRIPTION

The present invention is based, in part, on newly identified markerswhich are over- or under-abundant in samples, such as blood serum, frompatients who have endometriosis as compared to unaffected controlsamples, e.g., serum samples from control subjects that do not haveendometriosis. The altered abundance of one or more of these markers ina patient sample is herein correlated with the endometriosis. An alteredabundance of some of these markers is also correlated with the stage andclinical outcome of the patient. The invention provides compositions,kits, and methods for assessing the stage of the endometriosis, as wellas for treating patients afflicted with endometriosis.

The compositions, kits, and methods of the invention have the followinguses, among others:

-   -   1) assessing whether a patient is afflicted with endometriosis;    -   2) assessing the stage of endometriosis in a human patient;    -   3) predicting the clinical outcome of a patient diagnosed with        endometriosis;    -   4) assessing the nature of endometriosis in a patient;    -   5) making antibodies, antibody fragments or antibody derivatives        that are useful for assessing whether a patient is afflicted        with endometriosis;    -   6) assessing the efficacy of one or more test compounds for        inhibiting endometriosis in a patient;    -   7) assessing the efficacy of a therapy for inhibiting        endometriosis in a patient;    -   8) monitoring the progression of endometriosis in a patient; and    -   9) selecting a composition or therapy for inhibiting        endometriosis in a patient.

The invention thus includes a method of assessing whether a patient isafflicted with endometriosis. This method comprises comparing theabundance of a marker of the invention (listed in Table 1) in a patientsample and the normal abundance of the marker. An altered abundance ofthe marker in the patient sample as compared to the normal abundance isan indication that the patient is afflicted with endometriosis.

The invention further includes polypeptides comprising the entirety, ora segment of any of the sequences of SEQ ID NO (AAs) and homologuesthereof. Gene delivery vehicles, host cells and compositions (alldescribed herein) containing nucleic acids encoding the polypeptides ofthe invention are also included.

As described herein, endometriosis in a patient is associated with analtered abundance of one or more markers of the invention in a sample ortissue obtained from the patient.

Any marker or combination of markers of the invention, as well as anyknown markers in combination with the markers of the invention, may beused in the compositions, kits, and methods of the present invention. Ingeneral, it is preferable to use markers for which the differencebetween the abundance of the marker in a sample from a patient havingendometriosis and the normal abundance of the same marker is as great aspossible. Although this difference can be as small as the limit ofdetection of the method for assessing abundance of the marker, it ispreferred that the difference be at least greater than the standarderror of the assessment method, and preferably a difference of at least2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold or greater than the normalabundance of the same marker.

It will be appreciated that a variety of patient samples may be used inthe methods of the present invention. In these embodiments, theabundance of the marker can be assessed by assessing the amount (e.g.absolute amount or concentration) of the marker in the patient sample,e.g., a bodily fluid, such as a serum sample. The bodily fluid can beobtained from the subject using any available method, which may beselected on the basis of the amount of fluid required. In certain cases,a collected blood sample may be used in a variety of tests and only aportion or aliquot of the sample drawn will be required for use in themethods described herein. The amount of the marker(s) of the inventioncan be determined in whole blood or in a fraction of the blood.Preferably, the amount of marker is determined for a cell-free fractionof the blood, such as the plasma or the serum. It is particularlypreferred to determine the amount of marker(s) in the serum. Inembodiments in which only a fraction of the blood is used in theanalysis, the method also includes the steps of separating the desiredblood fraction from the whole blood acquired from the subject. Thisseparation of blood fractions can be achieved using methods which arewell-known in the art.

The bodily fluid may be further processed, as is known in the art, priorto the measurement of the marker. For example, the fluid can beprocessed to remove a particular protein, such as serum albumin, a setof proteins, or cells or cell components which are present in the fluidand which may interfere with the analysis. The processing can includesteps such as precipitation, chromatography, centrifugation,ultrafiltration and dialysis.

The cell sample can, of course, be subjected to a variety of well-knownpost-collection preparative and storage techniques (e.g., nucleic acidand/or protein extraction, fixation, storage, freezing, ultrafiltration,concentration, evaporation, centrifugation, etc.) prior to assessing theamount of the marker in the sample.

In one embodiment the abundance of the marker(s) of the invention in thesample is determined using chromatography, such as liquid chromatographyor gas chromatography, with a suitable detection system. In oneembodiment, the chromatographic step separates the bodily fluid intofractions, and at least one of the fractions comprises the markerpolypeptide. The fraction comprising the marker can be identified using,for example, mass spectrometry, and the amount of marker present can bedetermined using methods which are well known in the art, for example,by comparing the ion current generated in the mass spectrometer for themarker to that generated by an internal standard of known concentration.

In another embodiment, the abundance of a marker is assessed using anantibody (e.g. a radio-labeled, chromophore-labeled,fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative(e.g. an antibody conjugated with a substrate or with the protein orligand of a protein-ligand pair {e.g. biotin-streptavidin}), or anantibody fragment (e.g. a single-chain antibody, an isolated antibodyhypervariable domain, etc.) which binds specifically with a markerprotein or fragment thereof, including a marker protein which hasundergone all or a portion of its normal post-translationalmodification.

Because the compositions, kits, and methods of the invention rely ondetection of a difference in the abundance of one or more markers of theinvention, it is preferable that the abundance of the marker issignificantly greater than the minimum detection limit of the methodused to assess abundance.

It is understood that by routine screening of additional patient samplesusing one or more of the markers of the invention, it will be realizedthat certain of the markers are associated with endometriosis of variousstages based on a weighted point system. The number, size, and locationof endometrial implants, plaques, endometriomas, and/or adhesions areconsidered. According to the American Society for Reproductive Medicinerevised classification of endometriosis, Stage I (minimal): 1-5; stage11 (mild): 6-15; stage III (moderate): 16-40; stage 1V (severe): >40(Revised ASRM classification. Fertil Steri 1997; 67:819.) In addition,as a greater number of patient samples are assessed for expression ofthe markers of the invention and the outcomes of the individual patientsfrom whom the samples were obtained are correlated, it will also beconfirmed that altered abundance of certain of the markers of theinvention is strongly correlated with endometriosis and otherendometriosis-related diseases or conditions. The compositions, kits,and methods of the invention are thus useful for characterizing thestage, and nature of endometriosis in patients.

When the compositions, kits, and methods of the invention are used forcharacterizing the stage and nature of endometriosis in a patient, it ispreferred that the marker or panel of markers of the invention isselected such that a positive result is obtained in at least about 20%,and preferably at least about 40%, 60%, or 80%, and more preferably insubstantially all patients afflicted with endometriosis of thecorresponding stage and nature.

When a plurality of markers of the invention are used in thecompositions, kits, and methods of the invention, the abundance of eachmarker in a patient sample can be compared with the normal abundance ofeach of the plurality of markers in normal samples of the same type,either in a single reaction mixture (i.e. using reagents, such asdifferent fluorescent probes, for each marker) or in individual reactionmixtures corresponding to one or more of the markers. In one embodiment,a significantly increased abundance of more than one of the plurality ofmarkers in the sample, relative to the corresponding normal levels, isan indication that the patient is afflicted with endometriosis. Inanother embodiment, a significantly lowered abundance of more than oneof the plurality of markers in the sample, relative to the correspondingnormal levels, is an indication that the patient is afflicted withendometriosis. In another embodiment, a significantly lowered abundanceof a first marker in the sample and a significantly increased abundanceof a second marker, relative to the corresponding normal levels, is anindication that the patient is afflicted with endometriosis. When aplurality of markers is used, it is preferred that 2, 3, 4, 5, 6, 7, 8,9, 10, 12, 15, 20, 30, or 50 or more individual markers be used. Ingeneral, it is preferable to use the fewest markers required to providea test with the desired characteristics.

In one preferred embodiment, a marker polypeptide derived from each oftwo or more parent proteins can be monitored. For example, one set ofmarker polypeptides which can be monitored includesH-ADSGEGDFLAEGGGVR-OH (SEQ ID NO:22); Pyr-EGVNDNEEGFFSA-OH (SEQ IDNO:28); H-HWESASLL-OH (SEQ ID NO:37); H-EETKENEGFTVTAEG-OH (SEQ IDNO:35); and H-TQEKNPLPSKETIEQEKQAGES-OH (SEQ ID NO:42).

Markers associated with the presence of endometriosis have beendescribed, including those disclosed in U.S. Pat. Nos. 5,843,673;5,618,689 and 6,525,187 and published PCT applications WO 00/43789; WO99/55902; WO 00/63675; WO 96/20404; WO 99/63116; WO 01/62959; WO95/13821; and WO 00/47739, the contents of all of which are incorporatedherein by reference. These markers are not, of course, included amongthe markers of the invention, although they may be used together withone or more markers of the invention in a panel of markers, for example.

It is recognized that the compositions, kits, and methods of theinvention will be of particular utility to patients having an enhancedrisk of developing endometriosis and their medical advisors. Patientsrecognized as having an enhanced risk of developing endometriosisinclude, for example, patients having a familial history ofendometriosis, patients identified as having altered abundance of amarker of the invention, and patients with a history of pelvic pain,abdominal pain, irregular menstruation and/or infertility.

The normal abundance of a marker in a particular type of biologicalsample (i.e., in samples from patients that do not have endometriosis)can be assessed in a variety of ways. In one embodiment, as furtherinformation becomes available as a result of routine performance of themethods described herein, population-average values for normal abundanceof the markers of the invention may be used. In other embodiments, thenormal abundance of a marker may be determined by assessing abundance ofthe marker in a patient sample obtained from anon-endometriosis-afflicted patient, from a patient sample obtained froma patient before the suspected onset of endometriosis in the patient,from archived patient samples, and the like.

The invention includes compositions, kits, and methods for assessing thepresence of endometriosis in a sample (e.g. an archived tissue sample ora sample obtained from a patient). These compositions, kits, and methodsare substantially the same as those described above, except that, wherenecessary, the compositions, kits, and methods are adapted for use withsamples other than patient samples. For example, when the sample to beused is a parafinized, archived human tissue sample, it can be necessaryto adjust the ratio of compounds in the compositions of the invention,in the kits of the invention, or the methods used to assess levels ofmarker expression in the sample. Such methods are well known in the artand within the skill of the ordinary artisan.

The invention includes a kit for assessing the presence of endometriosis(e.g. in a sample such as a patient sample). The kit comprises aplurality of reagents, each of which is capable of binding specificallywith a marker polypeptide. Suitable reagents for binding with a markerpolypeptide include antibodies, antibody derivatives, antibodyfragments, and the like.

The kit of the invention may optionally comprise additional componentsuseful for performing the methods of the invention. By way of example,the kit may comprise fluids (e.g. SSC buffer) suitable for binding anantibody with a polypeptide with which it specifically binds, one ormore sample compartments, instructional material, such as a instructionmanual, which describes performance of a method of the invention, and apositive and negative control.

The invention also includes a method of making an isolated hybridomawhich produces an antibody useful for assessing whether a patient isafflicted with endometriosis. In this method, a protein or polypeptidecomprising the entirety or a segment of a marker polypeptide issynthesized or isolated (e.g. by purification from a cell in which it isexpressed or by transcription and translation of a nucleic acid encodingthe protein or polypeptide in vivo or in vitro using known methods). Avertebrate, preferably a mammal such as a mouse, rat, rabbit, or sheep,is immunized using the protein or polypeptide. The vertebrate mayoptionally (and preferably) be immunized at least one additional timewith the protein or polypeptide, so that the vertebrate exhibits arobust immune response to the protein or polypeptide. Splenocytes areisolated from the immunized vertebrate and fused with an immortalizedcell line to form hybridomas, using any of a variety of methods wellknown in the art. Hybridomas formed in this manner are then screenedusing standard methods to identify one or more hybridomas which producean antibody which specifically binds with the marker protein or afragment thereof. The invention also includes hybridomas made by thismethod and antibodies made using such hybridomas.

The invention also includes a method of assessing the efficacy of a testcompound for inhibiting endometriosis. As described above, differencesin the abundance of the markers of the invention correlate withendometriosis. Although it is recognized that changes in the levels ofcertain of the markers of the invention likely result fromendometriosis, it is likewise recognized that changes in the abundanceof other of the markers of the invention may be directly or indirectlyassociated with the induction, maintenance, and/or promotion ofendometriosis. Thus, compounds which inhibit endometriosis in a patientwill cause the abundance of one or more of the markers of the inventionto change to a level nearer the normal abundance level for that marker.

This method thus comprises comparing abundance of a marker in a firstpatient sample and maintained in the presence of the test compound andexpression of the marker in a second patient sample and maintained inthe absence of the test compound. A significantly altered abundance of amarker of the invention in the presence of the test compound is anindication that the test compound inhibits endometriosis. The patientsamples may, for example, be aliquots of a single sample of normalendometrial cells obtained from a patient, pooled samples of normalendometrial cells obtained from a patient, cells of a normal endometrialcell line, aliquots of a single sample of endometrial cells obtainedfrom a patient, pooled samples of endometrial cells obtained from apatient, cells of an endometrial cell line, a serum sample, or the like.In one embodiment, the samples include serum from the patient orendometrial cells obtained from a patient and a plurality of compoundsknown to be effective for inhibiting endometriosis are tested in orderto identify the compound which is likely to best inhibit theendometriosis in the patient.

This method may likewise be used to assess the efficacy of a therapy forinhibiting endometriosis in a patient. In this method, the abundance ofone or more markers of the invention in a pair of samples (one subjectedto the therapy, the other not subjected to the therapy) is assessed. Aswith the method of assessing the efficacy of test compounds, if thetherapy induces a significantly altered abundance (i.e., causes theabundance to approach normal values) of a marker of the invention thenthe therapy is efficacious for inhibiting endometriosis. As above, ifsamples from a selected patient are used in this method, thenalternative therapies can be assessed in vitro in order to select atherapy most likely to be efficacious for inhibiting endometriosis inthe patient.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid molecules,including nucleic acids which encode a marker polypeptide or a portion(fragment) thereof. Isolated nucleic acids of the invention also includenucleic acid molecules sufficient for use as hybridization probes toidentify these nucleic acid molecules and fragments of these nucleicacid molecules, e.g., those suitable for use as PCR primers for theamplification or mutation of the nucleic acid molecules. As used herein,the term “nucleic acid molecule” is intended to include DNA molecules(e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogsof the DNA or RNA generated using nucleotide analogs. The nucleic acidmolecule can be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Preferably, an “isolated” nucleic acid moleculeis free of sequences (preferably protein-encoding sequences) whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kB, 4kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention can be isolated usingstandard molecular biology techniques and the sequence information inthe database records described herein. Using all or a portion of suchnucleic acid sequences, nucleic acid molecules of the invention can beisolated using standard hybridization and cloning techniques (e.g., asdescribed in Sambrook et al., ed., Molecular Cloning: A LaboratoryManual, 2nd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989).

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA, or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, nucleotidescorresponding to all or a portion of a nucleic acid molecule of theinvention can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which has a nucleotidesequence complementary to the nucleotide sequence of a marker nucleicacid or to the nucleotide sequence of a nucleic acid encoding a markerpolypeptide. A nucleic acid molecule which is complementary to a givennucleotide sequence is one which is sufficiently complementary to thegiven nucleotide sequence that it can hybridize to the given nucleotidesequence thereby forming a stable duplex.

Moreover, a nucleic acid molecule of the invention can comprise only aportion of a nucleic acid sequence, wherein the full length nucleic acidsequence comprises a marker nucleic acid or which encodes a markerpolypeptide. Such nucleic acids can be used, for example, as a probe orprimer. The probe/primer typically is used as one or more substantiallypurified oligonucleotides. The oligonucleotide typically comprises aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 7, preferably about 15, more preferably about 25, 50,75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutivenucleotides of a nucleic acid of the invention.

Probes based on the sequence of a nucleic acid molecule of the inventioncan be used to detect transcripts or genomic sequences corresponding toone or more markers of the invention. The probe comprises a label groupattached thereto, e.g., a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as part of adiagnostic test kit for identifying cells or tissues which mis-expressthe polypeptide, such as by measuring levels of a nucleic acid moleculeencoding the polypeptide in a sample of cells from a subject, e.g.,detecting mRNA levels or determining whether a gene encoding thepolypeptide has been mutated or deleted.

It will be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequence can existwithin a population (e.g., the human population). Such geneticpolymorphisms can exist among individuals within a population due tonatural allelic variation. An allele is one of a group of genes whichoccur alternatively at a given genetic locus. In addition, it will beappreciated that DNA polymorphisms that affect RNA expression levels canalso exist that may affect the overall expression level of that gene(e.g., by affecting regulation or degradation).

As used herein, the phrase “allelic variant” refers to a nucleotidesequence which occurs at a given locus or to a polypeptide encoded bythe nucleotide sequence.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a polypeptidecorresponding to a marker of the invention. Such natural allelicvariations can typically result in 1-5% variance in the nucleotidesequence of a given gene. Alternative alleles can be identified bysequencing the gene of interest in a number of different individuals.This can be readily carried out by using hybridization probes toidentify the same genetic locus in a variety of individuals. Any and allsuch nucleotide variations and resulting amino acid polymorphisms orvariations that are the result of natural allelic variation and that donot alter the functional activity are intended to be within the scope ofthe invention.

In another embodiment, an isolated nucleic acid molecule of theinvention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250,300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600,1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, or morenucleotides in length and hybridizes under stringent conditions to a toa nucleic acid encoding a marker polypeptide. As used herein, the term“hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% (65%, 70%, preferably 75%) identical to eachother typically remain hybridized to each other. Such stringentconditions are known to those skilled in the art and can be found insections 6.3.1-6.3.6 of Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989). A preferred, non-limiting example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C.

In addition to naturally-occurring allelic variants of a nucleic acidmolecule of the invention that can exist in the population, the skilledartisan will further appreciate that sequence changes can be introducedby mutation thereby leading to changes in the amino acid sequence of theencoded polypeptide, without altering the biological activity of thepolypeptide encoded thereby. For example, one can make nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence without altering thebiological activity, whereas an “essential” amino acid residue isrequired for biological activity. For example, amino acid residues thatare not conserved or only semi-conserved among homologs of variousspecies may be non-essential for activity and thus would be likelytargets for alteration. Alternatively, amino acid residues that areconserved among the homologs of various species (e.g., murine and human)may be essential for activity and thus would not be likely targets foralteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding a variant marker polypeptide that contains changes inamino acid residues that are not essential for activity. Such variantmarker polypeptides differ in amino acid sequence from thenaturally-occurring marker polypeptides, yet retain biological activity.In one embodiment, such a variant marker polypeptide has an amino acidsequence that is at least about 40% identical, 50%, 60%, 70%, 80%, 90%,95%, or 98% identical to the amino acid sequence of a markerpolypeptide.

An isolated nucleic acid molecule encoding a variant marker polypeptidecan be created by introducing one or more nucleotide substitutions,additions or deletions into the nucleotide sequence of marker nucleicacids, such that one or more amino acid residue substitutions,additions, or deletions are introduced into the encoded polypeptide.Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded polypeptide can be expressed recombinantly andthe activity of the polypeptide can be determined.

II. Isolated Polypeptides and Antibodies

One aspect of the invention pertains to isolated marker polypeptides andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise antibodies directed against amarker polypeptide or a fragment thereof. In one embodiment, the nativemarker polypeptide can be isolated from cells or tissue sources by anappropriate purification scheme using standard polypeptide purificationtechniques. In another embodiment, a polypeptide comprising the whole ora segment of the marker polypeptide is produced by recombinant DNAtechniques. Alternative to recombinant expression, such a polypeptidecan be synthesized chemically using standard polypeptide synthesistechniques.

An “isolated” or “purified” polypeptide or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which thepolypeptide is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of polypeptide in whichthe polypeptide is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. Thus, polypeptide thatis substantially free of cellular material includes preparations ofpolypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight)of heterologous polypeptide (also referred to herein as a “contaminatingpolypeptide”). When the polypeptide or biologically active portionthereof is recombinantly produced, it is also preferably substantiallyfree of culture medium, i.e., culture medium represents less than about20%, 10%, or 5% of the volume of the polypeptide preparation. When thepolypeptide is produced by chemical synthesis, it is preferablysubstantially free of chemical precursors or other chemicals, i.e., itis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the polypeptide. Accordingly suchpreparations of the polypeptide have less than about 30%, 20%, 10%, 5%(by dry weight) of chemical precursors or compounds other than thepolypeptide of interest.

Preferred marker polypeptides are those having an amino acid sequence ofany of the SEQ ID NO (AAs). Other useful polypeptides are substantiallyidentical (e.g., at least about 40%, preferably 50%, 60%, 70%, 80%, 90%,95%, or 99%) to one of these sequences and retain the functionalactivity of the corresponding naturally-occurring marker polypeptide yetdiffer in amino acid sequence due to natural allelic variation ormutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the BLASTN and BLASTX programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the BLASTN program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST polypeptide searches can be performed with the BLASTPprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a polypeptide molecules of the invention. To obtain gappedalignments for comparison purposes, a newer version of the BLASTalgorithm called Gapped BLAST can be utilized as described in Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402, which is able to performgapped local alignments for the programs BLASTN, BLASTP and BLASTX.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., BLASTX and BLASTN) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Yet another useful algorithm for identifying regions of local sequencesimilarity and alignment is the FASTA algorithm as described in Pearsonand Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. When usingthe FASTA algorithm for comparing nucleotide or amino acid sequences, aPAM120 weight residue table can, for example, be used with a k-tuplevalue of 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

The invention also provides polypeptides which can be used as internalstandards for the marker polypeptides of the invention, for example, inmethods employing mass spectrometry. Such polypeptides includepolypeptides expected to behave in the analysis substantially similar tothe marker, but that have a mass which is distinct from the marker. Suchpolypeptides include isotopically-labelled versions of the markerpolypeptides, including versions of the marker polypeptides whichinclude one or more deuterium, tritium, ¹⁵N, ¹³C, ¹⁴C, ³²P, ³⁵S or acombination thereof. Other suitable polypeptides for use as internalstandards include those differing from the marker polypeptides by asmall structural element, for example, the addition or deletion of amethylene (—CH₂—) group, a methyl group, or a halogen atom, such as afluorine, chlorine, bromine or iodine atom. Included are polypeptideswhich are highly homologous to one of the marker polypeptides, forexample, those differing by the identity of one amino acid residue fromthe marker sequence. Suitable amino acid substitutions includeconservative substitutions, including substitutions with homologousamino acid residues, such as substitution of praline with homoproline,substitutions among valine and leucine or isoleucine, and others as canbe determined by the skilled artisan.

In another embodiment, the invention relates to chimeric or fusionproteins comprising a marker polypeptide of the invention or a segmentthereof. As used herein, a “chimeric protein” or “fusion protein”comprises all or part (preferably a biologically active part) of amarker polypeptide operably linked to a heterologous polypeptide (i.e.,a polypeptide other than the marker polypeptide). Within the fusionprotein, the term “operably linked” is intended to indicate that themarker polypeptide or segment thereof and the heterologous polypeptideare fused in-frame to each other. The heterologous polypeptide can befused to the amino-terminus or the carboxyl-terminus of the markerpolypeptide or segment.

One useful fusion protein is a GST fusion protein in which a markerpolypeptide or segment is fused to the carboxyl terminus of GSTsequences. Such fusion proteins can facilitate the purification of arecombinant polypeptide of the invention.

In another embodiment, the fusion protein contains a heterologous signalsequence at its amino terminus. For example, the native signal sequenceof a marker polypeptide can be removed and replaced with a signalsequence from another protein. For example, the gp67 secretory sequenceof the baculovirus envelope protein can be used as a heterologous signalsequence (Ausubel et al., ed., Current Protocols in Molecular Biology,John Wiley & Sons, NY, 1992). Other examples of eukaryotic heterologoussignal sequences include the secretory sequences of melittin and humanplacental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yetanother example, useful prokaryotic heterologous signal sequencesinclude the phoA secretory signal (Sambrook et al., supra) and theprotein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).

Chimeric and fusion proteins of the invention can be produced bystandard recombinant DNA techniques. In another embodiment, the fusiongene can be synthesized by conventional techniques including automatedDNA synthesizers. Alternatively, PCR amplification of gene fragments canbe carried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (see,e.g., Ausubel et al., supra). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A nucleic acid encoding a polypeptide of the invention canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the polypeptide of the invention.

A signal sequence can be used to facilitate secretion and isolation ofmarker polypeptides. Signal sequences are typically characterized by acore of hydrophobic amino acids which are generally cleaved from themature protein during secretion in one or more cleavage events. Suchsignal polypeptides contain processing sites that allow cleavage of thesignal sequence from the mature proteins as they pass through thesecretory pathway. Thus, the invention pertains to marker polypeptides,fusion proteins or segments thereof having a signal sequence, as well asto such proteins from which the signal sequence has been proteolyticallycleaved (i.e., the cleavage products). In one embodiment, a nucleic acidsequence encoding a signal sequence can be operably linked in anexpression vector to a protein of interest, such as a marker polypeptideor a segment thereof. The signal sequence directs secretion of theprotein, such as from a eukaryotic host into which the expression vectoris transformed, and the signal sequence is subsequently or concurrentlycleaved. The protein can then be readily purified from the extracellularmedium by art recognized methods. Alternatively, the signal sequence canbe linked to the protein of interest using a sequence which facilitatespurification, such as with a GST domain.

Another aspect of the invention pertains to antibodies directed againsta polypeptide of the invention. In preferred embodiments, the antibodiesspecifically bind a marker polypeptide or a fragment thereof. The terms“antibody” and “antibodies”, as used interchangeably herein, refer toimmunoglobulin molecules as well as fragments and derivatives thereofthat comprise an immunologically active portion of an immunoglobulinmolecule, (i.e., such a portion contains an antigen binding site whichspecifically binds an antigen, such as a marker polypeptide, e.g., anepitope of a marker protein). An antibody which specifically binds to apolypeptide of the invention is an antibody which binds the polypeptide,but does not substantially bind other molecules in a sample, e.g., abiological sample, which naturally contains the polypeptide. Examples ofan immunologically active portion of an immunoglobulin molecule include,but are not limited to, single-chain antibodies (scAb), F(ab) andF(ab′)₂ fragments.

An isolated polypeptide of the invention or a fragment thereof can beused as an immunogen to generate antibodies. The full-length polypeptidecan be used or, alternatively, the invention provides antigenicpolypeptide fragments for use as immunogens. The antigenic polypeptideof the invention comprises at least 7 (preferably 8, 10, 15, 20, or 30or more) amino acid residues of the amino acid sequence of one of theproteins of the invention, and encompasses at least one epitope of theprotein such that an antibody raised against the polypeptide forms aspecific immune complex with the protein. Preferred epitopes encompassedby the antigenic polypeptide are regions that are located on the surfaceof the protein, e.g., hydrophilic regions. Hydrophobicity sequenceanalysis, hydrophilicity sequence analysis, or similar analyses can beused to identify hydrophilic regions. In preferred embodiments, anisolated marker polypeptide or fragment thereof is used as an immunogen.

An immunogen typically is used to prepare antibodies by immunizing asuitable (i.e. immunocompetent) subject such as a rabbit, goat, mouse,or other mammal or vertebrate. An appropriate immunogenic preparationcan contain, for example, recombinantly-expressed orchemically-synthesized polypeptide or polypeptide. The preparation canfurther include an adjuvant, such as Freund's complete or incompleteadjuvant, or a similar immunostimulatory agent. Preferred immunogencompositions are those that contain no other human proteins such as, forexample, immunogen compositions made using a non-human host cell forrecombinant expression of a polypeptide of the invention. In such amanner, the resulting antibody compositions have reduced or no bindingof human proteins other than a polypeptide of the invention.

The invention provides polyclonal and monoclonal antibodies. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope. Preferred polyclonal and monoclonal antibodycompositions are ones that have been selected for antibodies directedagainst a polypeptide of the invention. Particularly preferredpolyclonal and monoclonal antibody preparations are ones that containonly antibodies directed against a marker polypeptide or fragmentthereof.

Polyclonal antibodies can be prepared by immunizing a suitable subjectwith a polypeptide of the invention as an immunogen The antibody titerin the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized polypeptide. At an appropriate time afterimmunization, e.g., when the specific antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies (mAb) by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497, the human B cell hybridoma technique (see Kozbor etal., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see Coleet al., pp. 77-96 In Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., 1985) or trioma techniques. The technology for producinghybridomas is well known (see generally Current Protocols in Immunology,Coligan et al. ed., John Wiley & Sons, New York, 1994). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindthe polypeptide of interest, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody directed against a polypeptide of the invention canbe identified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withthe polypeptide of interest. Kits for generating and screening phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZ4P Phage Display Kit, Catalog No. 240612). Additionally,examples of methods and reagents particularly amenable for use ingenerating and screening antibody display library can be found in, forexample, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCTPublication No. WO 91/17271; PCT Publication No. WO 92/20791; PCTPublication No. WO 92/15679; PCT Publication No. WO 93/01288; PCTPublication No. WO 92/01047; PCT Publication No. WO 92/09690; PCTPublication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J.12:725-734.

The invention also provides recombinant antibodies that specificallybind a polypeptide of the invention. In preferred embodiments, therecombinant antibodies specifically binds a marker polypeptide orfragment thereof. Recombinant antibodies include, but are not limitedto, chimeric and humanized monoclonal antibodies, comprising both humanand non-human portions, single-chain antibodies and multi-specificantibodies. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567;and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated hereinby reference in their entirety.) Single-chain antibodies have an antigenbinding site and consist of a single polypeptides. They can be producedby techniques known in the art, for example using methods described inLadner et. al U.S. Pat. No. 4,946,778 (which is incorporated herein byreference in its entirety); Bird et al., (1988) Science 242:423-426;Whitlow et al., (1991) Methods in Enzymology 2:1-9; Whitlow et al.,(1991) Methods in Enzymology 2:97-105; and Huston et al., (1991) Methodsin Enzymology Molecular Design and Modeling: Concepts and Applications203:46-88. Multi-specific antibodies are antibody molecules having atleast two antigen-binding sites that specifically bind differentantigens. Such molecules can be produced by techniques known in the art,for example using methods described in Segal, U.S. Pat. No. 4,676,980(the disclosure of which is incorporated herein by reference in itsentirety); Holliger et al., (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Whitlow et al., (1994) Protein Eng. 7:1017-1026 and U.S.Pat. No. 6,121,424.

The antibodies of the invention can be isolated after production (e.g.,from the blood or serum of the subject) or synthesis and furtherpurified by well-known techniques. For example, IgG antibodies can bepurified using protein A chromatography. Antibodies specific for apolypeptide of the invention can be selected or (e.g., partiallypurified) or purified by, e.g., affinity chromatography. For example, arecombinantly expressed and purified (or partially purified) polypeptideof the invention is produced as described herein, and covalently ornon-covalently coupled to a solid support such as, for example, achromatography column. The column can then be used to affinity purifyantibodies specific for the polypeptides of the invention from a samplecontaining antibodies directed against a large number of differentepitopes, thereby generating a substantially purified antibodycomposition, i.e., one that is substantially free of contaminatingantibodies. By a substantially purified antibody composition is meant,in this context, that the antibody sample contains at most only 30% (bydry weight) of contaminating antibodies directed against epitopes otherthan those of the desired polypeptide of the invention, and preferablyat most 20%, yet more preferably at most 10%, and most preferably atmost 5% (by dry weight) of the sample is contaminating antibodies. Apurified antibody composition means that at least 99% of the antibodiesin the composition are directed against the desired polypeptide of theinvention.

An antibody directed against a polypeptide of the invention can be usedto isolate the polypeptide by standard techniques, such as affinitychromatography or immunoprecipitation. Moreover, such an antibody can beused to detect the marker polypeptide or fragment thereof (e.g., in acellular lysate or cell supernatant) in order to evaluate the level andpattern of expression of the marker. The antibodies can also be useddiagnostically to monitor polypeptide levels in tissues or body fluids(e.g. in an endometrial-associated body fluid) as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by the use of anantibody derivative, which comprises an antibody of the inventioncoupled to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Accordingly, in one aspect, the invention provides substantiallypurified antibodies, antibody fragments and derivatives, all of whichspecifically bind to a polypeptide of the invention and preferably, amarker polypeptide. In various embodiments, the substantially purifiedantibodies of the invention, or fragments or derivatives thereof, can behuman, non-human, chimeric and/or humanized antibodies. In anotheraspect, the invention provides non-human antibodies, antibody fragmentsand derivatives, all of which specifically bind to a polypeptide of theinvention and preferably, a marker polypeptide. Such non-humanantibodies can be goat, mouse, sheep, horse, chicken, rabbit, or ratantibodies. Alternatively, the non-human antibodies of the invention canbe chimeric and/or humanized antibodies. In addition, the non-humanantibodies of the invention can be polyclonal antibodies or monoclonalantibodies. In still a further aspect, the invention provides monoclonalantibodies, antibody fragments and derivatives, all of whichspecifically bind to a polypeptide of the invention and preferably, amarker polypeptide. The monoclonal antibodies can be human, humanized,chimeric and/or non-human antibodies.

The invention also provides a kit containing an antibody of theinvention conjugated to a detectable substance, and instructions foruse. Still another aspect of the invention is a pharmaceuticalcomposition comprising an antibody of the invention and apharmaceutically acceptable carrier. In one embodiment, thepharmaceutical composition comprises an antibody of the invention, atherapeutic moiety, and a pharmaceutically acceptable carrier.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a markerpolypeptide (or a portion of such a polypeptide). As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors,namely expression vectors, are capable of directing the expression ofgenes to which they are operably linked. In general, expression vectorsof utility in recombinant DNA techniques are often in the form ofplasmids (vectors). However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Methods in Enzymology: GeneExpression Technology vol. 185, Academic Press, San Diego, Calif.(1991). Regulatory sequences include those which direct constitutiveexpression of a nucleotide sequence in many types of host cell and thosewhich direct expression of the nucleotide sequence only in certain hostcells (e.g., tissue-specific regulatory sequences). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of polypeptide desired,and the like. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or polypeptides, includingfusion proteins or polypeptides, encoded by nucleic acids as describedherein.

The recombinant expression vectors of the invention can be designed forexpression of a marker polypeptide or a segment thereof in prokaryotic(e.g., E. coli) or eukaryotic cells (e.g., insect cells {usingbaculovirus expression vectors}, yeast cells or mammalian cells).Suitable host cells are discussed further in Goeddel, supra.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studieret al., p. 60-89, In Gene Expression Technology: Methods in Enzymologyvol. 185, Academic Press, San Diego, Calif., 1991). Target geneexpression from the pTrc vector relies on host RNA polymerasetranscription from a hybrid trp-lac fusion promoter. Target geneexpression from the pET 11d vector relies on transcription from a T7gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase(T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) orHMS174(DE3) from a resident prophage harboring a T7 gn1 gene under thetranscriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, p. 119-128,In Gene Expression Technology: Methods in Enzymology vol. 185, AcademicPress, San Diego, Calif., 1990. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., 1992, Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjanand Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987,Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpPicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.,1983, Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow andSummers, 1989, Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840)and pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., supra.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell(e.g., insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker willsurvive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce a marker polypeptide or asegment thereof. Accordingly, the invention further provides methods forproducing a marker polypeptide or a segment thereof using the host cellsof the invention. In one embodiment, the method comprises culturing thehost cell of the invention (into which a recombinant expression vectorencoding a marker polypeptide or a segment thereof has been introduced)in a suitable medium such that the polypeptide is produced. In anotherembodiment, the method further comprises isolating the markerpolypeptide or a segment thereof from the medium or the host cell.

IV. Pharmaceutical Compositions

The invention also provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., polypeptides, peptidomimetics, peptoids,small molecules or other drugs) which have a modulatory effect on theabundance of the marker.

The test compounds of the present invention may be obtained from anyavailable source, including systematic libraries of natural and/orsynthetic compounds. Test compounds may also be obtained by any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of polypeptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann et al., 1994,J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to polypeptidelibraries, while the other four approaches are applicable topolypeptide, non-peptide oligomer or small molecule libraries ofcompounds (Lam, 1997, Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/orspores, (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al., 1992,Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990,Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al.,1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol.222:301-310; Ladner, supra.).

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a polypeptide encoded by orcorresponding to a marker or biologically active portion thereof. Inanother embodiment, the invention provides assays for screeningcandidate or test compounds which bind to a polypeptide encoded by orcorresponding to a marker or biologically active portion thereof.Determining the ability of the test compound to directly bind to apolypeptide can be accomplished, for example, by coupling the compoundwith a radioisotope or enzymatic label such that binding of the compoundto the marker can be determined by detecting the labeled marker compoundin a complex. For example, compounds (e.g., marker substrates) can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, assay components can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In another embodiment, the invention provides assays for screeningcandidate or test compounds which modulate the expression of a marker

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a marker polypeptide can befurther confirmed in vivo, e.g., in a whole animal model for cellulartransformation and/or tumorigenesis.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a marker modulating agent) can be used in ananimal model to determine the efficacy, toxicity, or side effects oftreatment with such an agent. Alternatively, an agent identified asdescribed herein can be used in an animal model to determine themechanism of action of such an agent. Furthermore, this inventionpertains to uses of novel agents identified by the above-describedscreening assays for treatments as described herein.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Predictive Medicine

The present invention pertains to the field of predictive medicine inwhich diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningthe level of expression of one or more marker polypeptides in order todetermine whether an individual is at risk of developing endometriosis.Such assays can be used for prognostic or predictive purposes to therebyprophylactically treat an individual prior to the onset ofendometriosis.

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other compounds administered either to inhibitendometriosis or to treat or prevent any other endometriosis-relateddisease, disorder or condition) on the abundance of a marker of theinvention in clinical trials. These and other agents are described infurther detail in the following sections.

A. Diagnostic Assays

An exemplary method for detecting the presence, absence or abundance ofa marker polypeptide in a biological sample involves obtaining abiological sample (e.g. an endometrial-associated body fluid, blood,blood plasma or serum) from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting the polypeptide.In vitro techniques for detection of a marker polypeptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence.

A general principle of such diagnostic and prognostic assays involvespreparing a sample or reaction mixture that may contain a marker, and aprobe, under appropriate conditions and for a time sufficient to allowthe marker and probe to interact and bind, thus forming a complex thatcan be removed and/or detected in the reaction mixture. These assays canbe conducted in a variety of ways.

For example, one method to conduct such an assay comprises anchoring themarker or probe onto a solid phase support, also referred to as asubstrate, and detecting target marker/probe complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, a sample from a subject, which is to be assayed for presenceand/or concentration of marker, can be anchored onto a carrier or solidphase support. In another embodiment, the reverse situation is possible,in which the probe can be anchored to a solid phase and a sample from asubject can be allowed to react as an unanchored component of the assay.

There are many established methods for anchoring assay components to asolid phase. These include, without limitation, marker or probemolecules which are immobilized through conjugation of biotin andstreptavidin. Such biotinylated assay components can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). In certain embodiments, the surfaces with immobilized assaycomponents can be prepared in advance and stored.

Other suitable carriers or solid phase supports for such assays includeany material capable of binding the class of molecule to which themarker or probe belongs. Well-known supports or carriers include, butare not limited to, glass, polystyrene, nylon, polypropylene, nylon,polyethylene, dextran, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite.

In order to conduct assays with the above mentioned approaches, thenon-immobilized component is added to the solid phase upon which thesecond component is anchored. After the reaction is complete,uncomplexed components may be removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized uponthe solid phase. The detection of marker/probe complexes anchored to thesolid phase can be accomplished in a number of methods outlined herein.

In a preferred embodiment, the probe, when it is the unanchored assaycomponent, can be labeled for the purpose of detection and readout ofthe assay, either directly or indirectly, with detectable labelsdiscussed herein and which are well-known to one skilled in the art.

It is also possible to directly detect marker/probe complex formationwithout further manipulation or labeling of either component (marker orprobe), for example by utilizing the technique of fluorescence energytransfer (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169;Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore labelon the first, ‘donor’ molecule is selected such that, upon excitationwith incident light of appropriate wavelength, its emitted fluorescentenergy will be absorbed by a fluorescent label on a second ‘acceptor’molecule, which in turn is able to fluoresce due to the absorbed energy.Alternately, the ‘donor’ polypeptide molecule may simply utilize thenatural fluorescent energy of tryptophan residues. Labels are chosenthat emit different wavelengths of light, such that the ‘acceptor’molecule label may be differentiated from that of the ‘donor’. Since theefficiency of energy transfer between the labels is related to thedistance separating the molecules, spatial relationships between themolecules can be assessed. In a situation in which binding occursbetween the molecules, the fluorescent emission of the ‘acceptor’molecule label in the assay should be maximal. An FET binding event canbe conveniently measured through standard fluorometric detection meanswell known in the art (e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe torecognize a marker can be accomplished without labeling either assaycomponent (probe or marker) by utilizing a technology such as real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S, andUrbaniczky, C., 1991, Anal. Chem. 63:2338-2345 and Szabo et al., 1995,Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or “surfaceplasmon resonance” is a technology for studying biospecific interactionsin real time, without labeling any of the interactants (e.g., BIAcore).Changes in the mass at the binding surface (indicative of a bindingevent) result in alterations of the refractive index of light near thesurface (the optical phenomenon of surface plasmon resonance (SPR)),resulting in a detectable signal which can be used as an indication ofreal-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic andprognostic assays can be conducted with marker and probe as solutes in aliquid phase. In such an assay, the complexed marker and probe areseparated from uncomplexed components by any of a number of standardtechniques, including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, marker/probe complexes may be separated from uncomplexedassay components through a series of centrifugal steps, due to thedifferent sedimentation equilibria of complexes based on their differentsizes and densities (see, for example, Rivas, G., and Minton, A. P.,1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographictechniques may also be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format, for example, the relativelylarger complex may be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of themarker/probe complex as compared to the uncomplexed components may beexploited to differentiate the complex from uncomplexed components, forexample through the utilization of ion-exchange chromatography resins.Such resins and chromatographic techniques are well known to one skilledin the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. Winter11(1-6):141-8; Hage, D. S., and Tweed, S. A. J Chromatogr B Biomed SciAppl 1997 Oct. 10; 699(1-2):499-525). Gel electrophoresis may also beemployed to separate complexed assay components from unbound components(see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology,John Wiley & Sons, New York, 1987-1999). In this technique, polypeptideor nucleic acid complexes are separated based on size or charge, forexample. In order to maintain the binding interaction during theelectrophoretic process, non-denaturing gel matrix materials andconditions in the absence of reducing agent are typically preferred.Appropriate conditions to the particular assay and components thereofwill be well known to one skilled in the art.

Alternatively, the abundance can be provided as a relative abundance. Todetermine a relative abundance of a marker, the abundance of the markeris determined for 10 or more samples of normal (non-endometriosis)samples, preferably 50 or more samples, prior to the determination ofthe abundance for the sample in question. The mean abundance of each ofthe markers assayed in the larger number of samples is determined andthis is used as a baseline abundance level for the marker. The abundanceof the marker determined for the test sample (absolute level ofexpression) is then divided by the mean abundance value obtained forthat marker. This provides a relative abundance.

Preferably, the samples used in the baseline determination will be fromblood, serum or plasma. The choice of the sample is dependent on the useof the relative abundance and the ease of obtaining and processing thesample. Using normal abundance as a mean abundance score aids invalidating whether the marker assayed is endometriosis specific (versusnormal). In addition, as more data is accumulated, the mean expressionvalue can be revised, providing improved relative abundance values basedon accumulated data. Abundance data provide a means for grading theseverity of the endometriosis state.

In another embodiment of the present invention, a marker polypeptide isdetected. A preferred agent for detecting a marker polypeptide of theinvention is an antibody capable of binding to the polypeptide or afragment thereof, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment or derivative thereof (e.g., Fab or F(ab′)₂) canbe used. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin.

A variety of formats can be employed to determine whether a samplecontains a polypeptide that binds to a given antibody. Examples of suchformats include, but are not limited to, enzyme immunoassay (EIA),radioimmunoassay (RIA), Western blot analysis and enzyme linkedimmunoabsorbant assay (ELISA). A skilled artisan can readily adapt knownpolypeptide/antibody detection methods for use in determining theabundance of a marker of the present invention within a given sample.

In one format, antibodies, or antibody fragments or derivatives, can beused in methods such as Western blots or immunofluorescence techniquesto detect the expressed polypeptides. In such uses, it is generallypreferable to immobilize either the antibody or polypeptides on a solidsupport. Suitable solid phase supports or carriers include any supportcapable of binding an antigen or an antibody. Well-known supports orcarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite.

One skilled in the art will know many other suitable carriers forbinding antibody or antigen, and will be able to adapt such support foruse with the present invention. For example, polypeptide isolated fromendometrial cells can be run on a polyacrylamide gel electrophoresis andimmobilized onto a solid phase support such as nitrocellulose. Thesupport can then be washed with suitable buffers followed by treatmentwith the detectably labeled antibody. The solid phase support can thenbe washed with the buffer a second time to remove unbound antibody. Theamount of bound label on the solid support can then be detected byconventional means.

In one embodiment, the abundance of a marker or collection of markers ofthe invention is determined using mass spectrometric methods. Forexample, following pretreatment, if any, the sample can be subjected toliquid chromatography/mass spectrometry (“LC/MS”), a method is which thecomponents of the sample are separated by the chromatographic step andthen analyzed by mass spectrometry. The method thus provides mass scandata as a function of retention time. Because of the possibility thatthe analyte of interest will co-elute with another sample componenthaving the same mass, the sample is preferably analyzed using liquidchromatography/mass spectrometry/mass spectrometry (“LC/MS/MS”), amethod in which the molecular ions from the first mass spectrometricstep are fragmented. Such a method typically provides one or morefragments which is unique to the analyte of interest and can bemonitored. The LC/MS/MS method thus provides a means of monitoring aspecific analyte without interference from other sample components andis particularly useful when the sample includes a complex mixture ofcomponents.

In one embodiment, the mass spectrometer is set to scan over a smallmass range which includes the mass of the analyte. In an LC/MS/MSmethod, the analyte can be determined using Selective ReactionMonitoring (“SRM”). In SRM, the parent mass of the analyte is specifiedfor further fragmentation, and a specific fragment ion is monitored. Ina Multiple Reaction Monitoring (“MRM”) experiment two or moreparent-fragment pairs are monitored.

In a preferred embodiment, a known quantity of internal standard isadded to the sample prior analysis of the sample. Preferably, aninternal standard is added for each marker polypeptide to be analyzed.The internal standard is preferably added to the sample prior to anyprocessing of the sample. The internal standard is a compound which isclosely related structurally to the analyte of interest, but that has adifferent mass than the analyte. For example, the internal standardshould be a compound which behaves essentially identically to the markerduring sample preparation, chromatographic separation and ionization inthe mass spectrometer. Thus, the internal standard is preferably, acompound which has structural, physical and chemical features which arevery close to those of the marker polypeptide. Suitable internalstandards for the present marker polypeptides include syntheticpolypeptides, such as synthetic, isotopically labeled polypeptideshaving the same structure, other than the isotopic label, as the markerpolypeptide to be analyzed. The internal standard polypeptides can beisototopically labeled in a suitable manner, as is known in the art,such as labeling with ²H (deuterium), ³H (tritium), ¹³C, ³³S, ³²P, ¹⁵Nand ¹⁷O. Preferably, the isotopic label is not a radioisotope. Preferredisotopic labels include ²H, ¹³C, ¹⁵N and ¹⁷O. The internal standard fora given polypeptide marker can also be a polypeptide which differsstructurally from the marker polypeptide in a minor way, such as theaddition or subtraction of a methylene group, methyl group, hydroxylgroup or halogen atom. For example, the following pairs of amino acidresidues can be substituted for each other in preparing an internalstandard for a given marker polypeptide: gycine/alanine; valine/leucine;valine/isoleucine; cysteine/homocysteine; asparagine/glutamine; asparticacid/glutamic acid; serine/threonine; and phenylalanine/tyrosine.Similar substitutions can be made using structurally related non-naturalamino acid residues, as is known in the art. Examples of suitableinternal standards for use in the present method include, but are notlimited to, polypeptides of the formula H-EETKENEGFTVTAEG-OH (d₈-Val)(SEQ ID NO:35) and Pyr-EGLNDNEEGFFSA-OH (SEQ ID NO:44).

Quantitation of a marker polypeptide of the invention can beaccomplished using methods known in the art. For example, for a givenmarker polypeptide, the synthetic standard can be used to generate acalibration curve. To generate a calibration curve, the syntheticstandard is added to blank or surrogate matrix in varying amounts, thesamples are pretreated using the desired protocol and then analyzed byLC/MS or LC/MS/MS. The area under the counts per second vs time curvefor the ion current (peak area) for the fragment (SIM, SRM) or each ofthe multiple fragments (MRM) is determined. A calibration curve isconstructed in which the concentration ratio of analyte to internalstandard is plotted versus peak area ratio. The peak area of thecorresponding fragment or fragments of the marker polypeptide isdetermined and the concentration of the marker polypeptide is foundusing the calibration curve.

The mass spectrometer to be used in the methods of the invention can be,for example, an ion trap mass spectrometer or a (triple)quadrupole massspectrometer. Electrospray (atmospheric pressure) ionization ispreferably used, but other methods for ionization can also be used, asis known in the art.

The invention also encompasses kits for detecting the presence of amarker polypeptide in a biological sample. Such kits can be used todetermine if a subject is suffering from or is at increased risk ofdeveloping endometriosis. For example, the kit can comprise a labeledcompound or agent capable of detecting one or more marker polypeptidesin a biological sample and means for determining the amount of thepolypeptide in the sample (e.g., an antibody which binds the polypeptideor fragment thereof.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to a markerpolypeptide; and, optionally, (2) a second, different antibody whichbinds to either the polypeptide or the first antibody and is conjugatedto a detectable label.

Kits to be used in mass spectrometric methods can include one or moresynthetic standards and/or one or more internal standards.

The kit can also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit can further comprise componentsnecessary for detecting the detectable label (e.g., an enzyme or asubstrate). The kit can also contain a control sample or a series ofcontrol samples which can be assayed and compared to the test sample.Each component of the kit can be enclosed within an individual containerand all of the various containers can be within a single package, alongwith instructions for interpreting the results of the assays performedusing the kit.

C. Monitoring Clinical Trials

Monitoring the influence of agents (e.g., drug compounds) on the levelof expression of a marker of the invention can be applied not only inbasic drug screening, but also in clinical trials. For example, theability of an agent to affect marker abundance can be monitored inclinical trials of subjects receiving treatment for endometriosis. In apreferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, polypeptide,nucleic acid, small molecule, or other drug candidate) comprising thesteps of (i) obtaining a pre-administration sample from a subject priorto administration of the agent; (ii) determining the abundance of one ormore selected markers of the invention in the pre-administration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) determining the abundance of the marker(s) in thepost-administration samples; (v) comparing the abundance of themarker(s) in the pre-administration sample with the abundance of themarker(s) in the post-administration sample or samples; and (vi)altering the administration of the agent to the subject accordingly. Forexample, increased abundance of certain marker polypeptide(s) during thecourse of treatment may indicate ineffective dosage and the desirabilityof increasing the dosage. Conversely, decreased expression of thesemarker polypeptide(s) may indicate efficacious treatment and no need tochange dosage.

D. Electronic Apparatus Readable Media and Arrays

Electronic apparatus readable media comprising a marker of the presentinvention is also provided. As used herein, “electronic apparatusreadable media” refers to any suitable medium for storing, holding orcontaining data or information that can be read and accessed directly byan electronic apparatus. Such media can include, but are not limited to:magnetic storage media, such as floppy discs, hard disc storage medium,and magnetic tape; optical storage media such as compact disc;electronic storage media such as RAM, ROM, EPROM, EEPROM and the like;general hard disks and hybrids of these categories such asmagnetic/optical storage media. The medium is adapted or configured forhaving recorded thereon a marker of the present invention.

As used herein, the term “electronic apparatus” is intended to includeany suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as a personal digital assistants(PDAs), cellular phone, pager and the like; and local and distributedprocessing systems.

As used herein, “recorded” refers to a process for storing or encodinginformation on the electronic apparatus readable medium. Those skilledin the art can readily adopt any of the presently known methods forrecording information on known media to generate manufactures comprisingthe markers of the present invention.

A variety of software programs and formats can be used to store themarker information of the present invention on the electronic apparatusreadable medium. For example, the marker nucleic acid sequence can berepresented in a word processing text file, formatted incommercially-available software such as WordPerfect and MicroSoft Word,or represented in the form of an ASCII file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like, as well as inother forms. Any number of data processor structuring formats (e.g.,text file or database) may be employed in order to obtain or create amedium having recorded thereon the markers of the present invention.

By providing the markers of the invention in readable form, one canroutinely access the marker sequence information for a variety ofpurposes. For example, one skilled in the art can use the nucleotide oramino acid sequences of the present invention in readable form tocompare a target sequence or target structural motif with the sequenceinformation stored within the data storage means. Search means are usedto identify fragments or regions of the sequences of the invention whichmatch a particular target sequence or target motif.

The present invention therefore provides a medium for holdinginstructions for performing a method for determining whether a subjecthas endometriosis or a pre-disposition to endometriosis, wherein themethod comprises the steps of determining the abundance of a marker and,based on the abundance of the marker, determining whether the subjecthas endometriosis or a pre-disposition to endometriosis and/orrecommending a particular treatment for endometriosis orpre-endometriosis condition.

The present invention further provides in an electronic system and/or ina network, a method for determining whether a subject has endometriosisor a pre-disposition to endometriosis associated with a marker whereinthe method comprises the steps of determining the abundance of themarker, and based on the abundance of the marker, determining whetherthe subject has endometriosis or a pre-disposition to endometriosis,and/or recommending a particular treatment for the endometriosis orpre-endometriosis condition. The method may further comprise the step ofreceiving phenotypic information associated with the subject and/oracquiring from a network phenotypic information associated with thesubject.

The present invention also provides in a network, a method fordetermining whether a subject has endometriosis or a pre-disposition toendometriosis associated with a marker, said method comprising the stepsof receiving information associated with the marker, receivingphenotypic information associated with the subject, acquiringinformation from the network corresponding to the marker and/orendometriosis, and based on one or more of the phenotypic information,the marker, and the acquired information, determining whether thesubject has endometriosis or a pre-disposition to endometriosis. Themethod may further comprise the step of recommending a particulartreatment for the endometriosis or pre-endometriosis condition.

The present invention also provides a business method for determiningwhether a subject has endometriosis or a pre-disposition toendometriosis, said method comprising the steps of receiving informationassociated with the marker, receiving phenotypic information associatedwith the subject, acquiring information from the network correspondingto the marker and/or endometriosis, and based on one or more of thephenotypic information, the marker, and the acquired information,determining whether the subject has endometriosis or a pre-dispositionto endometriosis. The method may further comprise the step ofrecommending a particular treatment for the endometriosis orpre-endometriosis condition.

E. Surrogate Markers

The markers of the invention may serve as surrogate markers for one ormore disorders or disease states or for conditions leading up to diseasestates, and in particular, endometriosis. As used herein, a “surrogatemarker” is an objective biochemical marker which correlates with theabsence or presence of a disease or disorder, or with the progression ofa disease or disorder (e.g., with the presence or absence of a tumor).The presence or quantity of such markers is independent of the disease.Therefore, these markers may serve to indicate whether a particularcourse of treatment is effective in lessening a disease state ordisorder. Surrogate markers are of particular use when the presence orextent of a disease state or disorder is difficult to assess throughstandard methodologies (e.g., early stage tumors), or when an assessmentof disease progression is desired before a potentially dangerousclinical endpoint is reached (e.g., an assessment of cardiovasculardisease may be made using cholesterol levels as a surrogate marker, andan analysis of HIV infection may be made using HIV RNA levels as asurrogate marker, well in advance of the undesirable clinical outcomesof myocardial infarction or fully-developed AIDS). Examples of the useof surrogate markers in the art include: Koomen et al. (2000) J. Mass.Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

VI. Experimental Protocol

A. Identification of the Markers of the Invention

Serum samples were obtained from women with endometriosis confirmed bylaparoscopy and from women having no complaints indicative ofendometriosis. Samples were also obtained from women diagnosed withpolycystic ovarian syndrome (PCOS) and pelvic inflammatory disease(PID). The serum samples were prepared for analysis and analyzed usingthe following protocol.

50 mL serum was diluted with 100 μL 500 μM aqueous glycine, pH 2.0. Thediluted serum was then ultrafiltered using a 10,000 dalton molecularweight cutoff membrane (Millipore; 14,000 RCF maximum) at 3,750 rpm on aswinging bucket for approximately 2.5 hours. The filtrate wastransferred to a container for HPLC analysis, which was performed usinga Grace Vydac C-8 column (0.21 cm×15 cm; Catalog No. 208TP5215), and aninjection volume of 0.1 mL. The column was eluted with a gradient of95/4.9/0.1 acetonitrile/water/formic acid from 2% to 60% over 30minutes. The eluate was analyzed by mass spectrometry using a Watersqudrupole-time-of-flight mass spectrometer with a Z-spray source and anmass/charge range of 200 to 2000 daltons. Quantitation of a particularretention time/mass to charge pair was taken as the ion count from themass spectrometer, which was assumed to be linear with the concentrationof the analyte.

The results for several of the markers of the invention are set forth inFIGS. 1 to 6, each of which is a scatter plot showing the concentrations(given as counts) of a given marker in the serum of women diagnosed withendometriosis, the serum of healthy women, and, in certain cases, theserum of women diagnosed with PCOS or PID. The data set forth in FIGS. 1and 2 indicate that the fibrinopeptide B derivative (SEQ ID NO:28), thefull length fibrinopeptide A (SEQ ID NO:3) and the fibrinopeptide Afragment (SEQ ID NO:7) are all present in higher concentrations in theserum of women diagnosed with endometriosis than in the serum of healthywomen. FIG. 3 indicates that the fibrinopeptide A derivative (SEQ IDNO:4) is present at higher concentrations in women diagnosed withendometriosis than in the serum of healthy women or women diagnosed withPCOS or PID. The data set forth in FIG. 4 demonstrate that the thymosinfragments (SEQ ID NOS:42 and 43) are present at lower concentrations inthe serum of women diagnosed with endometriosis, PCOS and PID than inthe serum of healthy women. FIG. 5 shows that phosphoserinefibrinopeptide A (SEQ ID NO:22) is present at lower concentrations inthe serum of healthy women than in the serum of women diagnosed withendometriosis, PCOS or PID. The data in FIG. 6 demonstrate that theinternal fibrinogen fragment (SEQ ID NO:29) is present at lowerconcentrations in the serum of women diagnosed with endometriosis thanin the serum of healthy women or women diagnosed with PCOS or PID.

B. Summary of the Tables

Tables 1-3 list markers of the invention obtained using the foregoingexperimental protocol. Table 1 lists all of the markers of theinvention, which are over- or under-expressed in patient samplescompared to normal (i.e., a sample from a patient that does not haveendometriosis) samples. Table 2 lists markers whose over-expression maybe correlated with endometriosis as compared to normal samples frompatients that do not have endometriosis. Table 3 lists markers whoseunder-expression may be correlated with endometriosis as compared tonormal samples from patients that do not have endometriosis.

The contents of all references, patents, published patent applications,and database records, cited throughout this application, are herebyincorporated by reference.

OTHER EMBODIMENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of assessing whether a patient is afflicted withendometriosis, the method comprising comparing: a) the abundance of oneor more marker polypeptides selected from the group consisting of: iC3b(SEQ ID NO:33), or a fragment thereof; SEETKENEGFTVTAEG (SEQ ID NO:34),or a fragment thereof; EETKENEGFTVTAEG (SEQ ID NO:35), or a fragmentthereof; SDKPDMAEMEKFDKSKLKKTETQEKNLPSKETIEQEKQAGES (thymosin beta 1)(SEQ ID NO:38), or a fragment thereof;SDKPDMAEIEKFDKPKLKKTETQEKNPLPSKETIEQEKQAGES (thymosin beta 3) (SEQ IDNO:39), or a fragment thereof; thymosin beta 4 (SEQ ID NO:40), or afragment thereof; SDKSDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES (thymosinbeta 6) (SEQ ID NO:41), or a fragment thereof; TQEKNPLPSKETIEQEKQAGES(SEQ ID NO:42), or a fragment thereof; and pyr-EKNPLPSKETIEQEKQAGES (SEQID NO:43), or a fragment thereof, in a patient sample, wherein thefragment thereof consists of 7-15 consecutive amino acid residues of themarker polypeptide, and b) the normal abundance of the one or more ofmarker polypeptides in a sample from a control subject not afflictedwith endometriosis, wherein a significantly lower abundance of one ormore of said marker polypeptides in the patient sample as compared tothe normal abundance of one or more of said marker polypeptidesindicates that the patient is afflicted with endometriosis.
 2. A methodof assessing whether a patient is afflicted with endometriosis, themethod comprising comparing: a) the abundance of each of a plurality ofmarker polypeptides independently selected from the group consisting of:iC3b (SEQ ID NO:33), or a fragment thereof; SEETKENEGFTVTAEG (SEQ IDNO:34), or a fragment thereof; EETKENEGFTVTAEG (SEQ ID NO:35), or afragment thereof; SDKPDMAEMEKFDKSKLKKTETQEKNLPSKETIEQEKQAGES (thymosinbeta 1) (SEQ ID NO:38), or a fragment thereof;SDKPDMAEIEKFDKPKLKKTETQEKNPLPSKETIEQEKQAGES (thymosin beta 3) (SEQ IDNO:39), or a fragment thereof; thymosin beta 4 (SEQ ID NO:40), or afragment thereof; SDKSDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES (thymosinbeta 6) (SEQ ID NO:41), or a fragment thereof; TQEKNPLPSKETIEQEKQAGES(SEQ ID NO:42), or a fragment thereof; and pyr-EKNPLPSKETIEQEKQAGES (SEQID NO:43), or a fragment thereof, in a patient sample, wherein thefragment thereof consists of 7-15 consecutive amino acid residues of themarker polypeptide, and b) the normal abundance of each of the pluralityof marker polypeptides in a sample obtained from a control subject notafflicted with endometriosis, wherein a significantly lower abundance ofone or more of said marker polypeptides in the patient sample ascompared to the normal abundance of one or more of said markerpolypeptides indicates that the patient is afflicted with endometriosis.3. The method of claim 1 or 2, wherein the sample comprises a fluidselected from the group consisting of blood fluids, a blood fraction,lymph, ascitic fluids, gynecological fluids, urine, peritoneal fluid,cerebrospinal fluid, and fluids collected by vaginal rinsing.
 4. Themethod of claim 1 or 2, wherein the sample is blood serum or bloodplasma.
 5. The method of claim 1 or 2, wherein the abundance of saidmarker polypeptides in the samples is assessed by a method comprisingthe step of detecting the presence in the samples of said markerpolypeptides or a fragment thereof.
 6. The method of claim 5, whereinthe presence of said marker polypeptide or fragment thereof is detectedusing a reagent which specifically binds with said marker polypeptide orfragment thereof, wherein said reagent is selected from the groupconsisting of an antibody, an antibody derivative, and an antibodyfragment.
 7. The method of claim 2, wherein the plurality comprises atleast three of the marker polypeptides.
 8. The method of claim 7,wherein the plurality comprises at least five of the markerpolypeptides.